Apparatus and method for detecting and identifying infectious agents

ABSTRACT

Solid phase methods for the identification of an analyte in a biological medium, such as a body fluid, using bioluminescence are provided. A chip designed for performing the method and detecting the bioluminescence is also provided. Methods employing biomineralization for depositing silicon on a matrix support are also provided. A synthetic synapse is also provided.

RELATED APPLICATIONS This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional appplication Ser. No. 60/037,675, filed Feb.11, 1997 and to U.S. Provisional application Ser. No. 60/033,745, filedDec. 12, 1996.

Certain subject matter in this application is related to subject matterin U.S. application Ser. No. 08/757,046, filed Nov. 25, 1996, to BruceBryan entitled “BIOLUMINESCENT NOVELTY ITEMS” (B), and to U.S.application Ser. No. 08/597,274, filed Feb. 6, 1996, to Bruce Bryan,entitled “BIOLUMINESCENT NOVELTY ITEMS”. This application is alsorelated to U.S. application Ser. No. 08/908,909, filed Aug. 8, 1997, toBruce Bryan entitled “DETECTION AND VISUALIZATION OF NEOPLASMS AND OTHERTISSUES” and to U.S. Provisional application Ser. No. 60/023,374, filedAug. 8, 1996, entitled “DETECTION AND VISUALIZATION OF NEOPLASMS ANDOTHER TISSUES”, and also to published International PCT application No.WO 97/29,319.

The subject matter of each of the above noted U.S. applications,provisional applications and International application is hereinincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods for the identification of ananalyte in a biological medium using bioluminescence. More particularly,a method is provided for diagnosing diseases employing a solid phasemethodology and a luciferase-luciferin bioluminescence generatingsystem. Methods employing biomineralization for depositing silicon on amatrix support are also provided herein.

BACKGROUND OF THE INVENTION Bioluminescence

Luminescence is a phenomenon in which energy is specifically channeledto a molecule to produce an excited state. Return to a lower energystate is accompanied by release of a photon (hy). Luminescence includesfluorescence, phosphorescence, chemiluminescence and bioluminescence.Bioluminescence is the process by which living organisms emit light thatis visible to other organisms. Luminescence may be represented asfollows:

A+B→X*+Y

X*→X+hv,

where X* is an electronically excited molecule and hy represents lightemission upon return of X* to a lower energy state. Where theluminescence is bioluminescence, creation of the excited state derivesfrom an enzyme catalyzed reaction. The color of the emitted light in abioluminescent (or chemiluminescent or other luminescent) reaction ischaracteristic of the excited molecule, and is independent from itssource of excitation and temperature.

An essential condition for bioluminescence is the use of molecularoxygen, either bound or free in the presence of a luciferase.Luciferases, are oxygenases, that act on a substrate, luciferin, in thepresence of molecular oxygen and transform the substrate to an excitedstate. Upon return to a lower energy level, energy is released in theform of light [for reviews see, e.g., McElroy et al. (1966) in MolecularArchitecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall,Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 inChemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. N.Y., pp.321-358; Hastings, J. W. in (1995) Cell Physiology:Source Book, N.Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis andLife in the Deep Sea, Johnson, Vantage Press, N.Y., see, esp. pp.50-56].

Though rare overall, bioluminescence is more common in marine organismsthan in terrestrial organisms. Bioluminescence has developed from asmany as thirty evolutionarily distinct origins and, thus, is manifestedin a variety of ways so that the biochemical and physiologicalmechanisms responsible for bioluminescence in different organisms aredistinct. Bioluminescent species span many genera and includemicroscopic organisms, such as bacteria [primarily marine bacteriaincluding Vibrio species], fungi, algae and dinoflagellates, to marineorganisms, including arthropods, mollusks, echinoderms, and chordates,and terrestrial organism including annelid worms and insects.

Bioluminescence, as well as other types of chemiluminescence, is usedfor quantitative determinations of specific substances in biology andmedicine. For example, luciferase genes have been cloned and exploitedas reporter genes in numerous assays, for many purposes. Since thedifferent luciferase systems have different specific requirements, theymay be used to detect and quantify a variety of substances. The majorityof commercial bioluminescence applications are based on firefly[Photinus pyralis] luciferase. One of the first and still widely usedassays involves the use of firefly luciferase to detect the presence ofATP. It is also used to detect and quantify other substrates orco-factors in the reaction. Any reaction that produces or utilizesNAD(H), NADP(H) or long chain aldehyde, either directly or indirectly,can be coupled to the light-emitting reaction of bacterial luciferase.

Another luciferase system that has been used commercially for analyticalpurposes is the Aequorin system. The purified jellyfish photoprotein,aequorin, is used to detect and quantify intracellular Ca²⁺ and itschanges under various experimental conditions. The Aequorin photoproteinis relatively small [˜20 kDa], nontoxic, and can be injected into cellsin quantities adequate to detect calcium over a large concentrationrange [3×10⁻⁷ to 10⁻⁴ M].

Because of their analytical utility, many luciferases and substrateshave been studied and well-characterized and are commercially available[e.g., firefly luciferase is available from Sigma, St. Louis, Mo., andBoehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantlyproduced firefly luciferase and other reagents based on this gene or foruse with this protein are available from Promega Corporation, Madison,Wis.; the aequorin photoprotein luciferase from jellyfish and luciferasefrom Renilla are commercially available from Sealite Sciences, Bogart,Ga.; coelenterazine, the naturally-occurring substrate for theseluciferases, is available from Molecular Probes, Eugene, OR]. Theseluciferases and related reagents are used as reagents for diagnostics,quality control, environmental testing and other such analyses.

Chips, Arrays and Microelectronics

Microelectronics, chip arrays and other solid phase spaciallyaddressable arrays have been been developed for use in diagnostics andother applications. At present, methods for detection of positiveresults are inadequate or inconvenient. There exists a need forimproved, particularly more rapid detection methods.

Therefore, it is an object herein to provide detection means andmethods.

SUMMARY OF THE INVENTION

A method is provided for diagnosing diseases, particularly infectiousdiseases, using chip methodology and a luciferase-luciferinbioluminescence generating system. A chip device for practicing themethods is also provided herein. The chip includes an integratedphotodetector that detects the photons emitted by the bioluminescencegenerating system. The method may be practiced with any suitable chipdevice, including self-addressable and non-self addressable formats,that is modified as described herein for detection of generated photonsby the bioluminescence generating systems. The chip device providedherein is adaptable for use in an array format for the detection andidentification of infectious agents in biological specimens.

To prepare the chip, a suitable matrix for chip production is selected,the chip is fabricated by suitably derivatizing the matrix for linkageof macromolecules, and including linkage of photodiodes,photomultipliers CCD (charge coupled device) or other suitable detector,for measuring light production; attaching an appropriate macromolecule,such as a biological molecule or anti-ligand, e.q., a receptor, such asan antibody, to the chip, preferably to an assigned location thereon.Photodiodes are presently among the preferred detectors, and specifiedherein. It is understood, however, that other suitable detectors may besubstituted therefor.

In one embodiment, the chip is made using an integrated circuit with anarray, such as an X-Y array, of photodetectors. The surface of circuitis treated to render it inert to conditions of the diagnostic assays forwhich the chip is intended, and is adapted, such as by derivatizationfor linking molecules, such as antibodies. A selected antibody or panelof antibodies, such as an antibody specific for particularly bacterialantigen, is affixed to the surface of the chip above each photodetector.After contacting the chip with a test sample, the chip is contacted asecond antibody linked to a component of a bioluminescence generatingsystem, such as a luciferase or luciferin, specific for the antigen. Theremaining components of the bioluminescence generating reaction areadded, and, if any of the antibodies linked to a component of abioluminescence generating system are present on the chip, light will begenerated and detected by the adjacent photodetector. The photodetectoris operatively linked to a computer, which is programmed withinformation identifying the linked antibodies, records the event, andthereby identifies antigens present in the test sample.

The chip is employed in any desired assay, such as an assay forinfectious disease or antibiotic sensitivity, by, for example, linkingan antibody or a panel of antibodies, to the surface, contacting thechip with a test sample of a body fluid, such as urine, blood andcerebral spinal fluid (CFS), for a sufficient time, depending upon assayformat, such as to bind the a target in the sample; washing the chip andthen incubating with a secondary antibody conjugated to a luciferase oran antibody:luciferase fusion protein; initiating the bioluminescentreaction; detecting light emitted at each location bound with a targetthrough the photodiode in the chip; transferring the electronic signalfrom the chip to a computer for analysis.

In one embodiment, the chip is a nonself-addressable, microelectronicdevice for detecting photons of light emitted by light-emitting chemicalreactions. The device includes a substrate, an array of loci, hereindesignated micro-locations, defined thereon, and an independentphotodetector optically coupled to each micro-location. Eachmicro-location holds a separate chemical reactant that will emit photonsof light when a reaction takes place thereat. Each photodetectorgenerates a sensed signal responsive to the photons emitted at thecorresponding micro-location when the reaction takes place thereat, andeach photodetector is independent from the other photodetectors. Thedevice also includes an electronic circuit that reads the sensed signalgenerated by each photodetector and generates output data signalstherefrom. The output data signals are indicative of the light emittedat each micro-location.

In another embodiment, a microelectronic device for detecting andidentifying analytes in a fluid sample using light-emitting reactions isprovided. The device includes a substrate, an array of micro-locationsdefined thereon for receiving the fluid sample to be analyzed, aseparate targeting agent attached to an attachment layer of eachmicro-location, and an independent photodetector optically coupled toeach micro-location. Each targeting agent is, preferably, specific forbinding a selected analyte that may be present in the received sample.Each photodetector generates a sensed signal responsive to photons oflight emitted at the corresponding micro-location when the selectedanalyte bound thereto is exposed to a secondary binding agent alsospecific for binding the selected analyte or the targetingagent-selected analyte complex and linked to one or more components of alight-emitting reaction. The chip is then reacted with the remainingcomponents to emit the photons when the selected analyte is present. Anelectronic circuit reads the sensed signal generated by eachphotodetector and generates output data signals therefrom that areindicative of the light emitted at each micro-location.

In yet another embodiment, a microelectronic device for detecting andidentifying analytes in a biological sample using luciferase-luciferinbio-luminescence is provided. The device includes a substrate, an arrayof micro-locations defined thereon for receiving the sample to beanalyzed, a separate anti-ligand, such as a receptor antibody, attachedto an attachment layer of each micro-location, and an independentphotodetector optically coupled to each micro-location. Each receptorantibody is specific for binding a selected analyte that may be presentin the received sample. Each photodetector generates a sensed signalresponsive to bioluminescence emitted at the correspondingmicro-location when the selected analyte bound to the correspondingreceptor antibody is exposed to a secondary antibody also specific tothe selected analyte or to the receptor antibody-selected analytecomplex and linked to one or more components of a luciferase-luciferinreaction, and is then reacted with the remaining components to generatethe bioluminescence when the selected analyte is present. An electroniccircuit reads the sensed signal from each photodetector and generatesoutput data signals therefrom. The output data signals are indicative ofthe bioluminescence emitted at each micro-location by the reaction.

In another embodiment, a method of detecting and identifying analytes ina biological sample using luciferase-luciferin bioluminescence isprovided. The method includes providing a microelectronic device havinga surface with an array of micro-locations defined thereon, derivatizingthe surface to permit or enhance the attachment of a receptor antibodyor plurality of antibodies thereto at each micro-location, and attachinga specific receptor antibody or plurality thereof to the surface at eachmicro-location. The selected antibody is specific for binding to aselected analyte that may be present in the sample. The method alsoincludes applying the sample to the surface such that the selectedanalytes will bind to the receptor antibody attached to the surface ateach micro-location, washing the sample from the surface after waiting asufficient period of time for the selected analytes to bind with thereceptor antibody at each micro-location, exposing the surface to asecondary antibody specific to bind the selected analyte already boundto the receptor antibody at each micro-location when the selectedanalyte is present, the secondary antibody linked to one of a luciferaseand a luciferin, and initiating the reaction by applying the other ofthe luciferase and luciferin to the surface. The method also includesdetecting photons of light emitted by the reaction using a photodetectoroptically coupled to each micro-location, each photodetector generatinga sensed signal representative of the bio-luminescent activity thereat,reading the sensed signal from each photodetector and generating outputdata signals therefrom indicative of the bioluminescence emitted at eachmicro-location by the reaction.

In a further embodiment, a system for detecting and identifying analytesin a biological sample using luciferase-luciferin bioluminescence isprovided. The system includes: a microelectronic device including anarray of micro-locations for receiving the sample; a separate receptorantibody attached to an attachment layer of each micro-location, eachreceptor antibody is specific for a selected analyte that may be presentin the received sample; a photodetector that generates a sensed signalresponsive to bioluminescence emitted at the correspondingmicro-location when the selected analyte bound to the correspondingreceptor antibody is exposed to a secondary antibody also specific tothe selected analyte and linked to one of a luciferase and a luciferin,and is then reacted with the other of the luciferase and luciferin togenerate the bioluminescence when the selected analyte is present, andan electronic circuit which reads the sensed signal from eachphotodetector and generates output data signals therefrom indicative ofthe bioluminescence emitted at each micro-location by the reaction. Thesystem includes a processing instrument including an input interfacecircuit for receiving the output data signals indicative of thebio-luminescence emitted at each micro-location, a memory circuit forstoring a data acquisition array having a location associated with eachmicro-location, an output device for generating visible indicia inresponse to an output device signal and a processing circuit. Theprocessing circuit reads the output data signals received by the inputinterface circuit, correlates these signals with the correspondingmicro-locations, integrates the correlated output data signals for adesired time period by accumulating them in the data acquisition array,and generates the output device signal which, when applied to the outputdevice, causes the output device to generate visible indicia related tothe presence of the selected analytes.

In other embodiments, the chip is self-addressable. When usingself-addressable chips in the method, presently preferred are thoseadaptable to microelectronic self addressable, self-assembling chips andsystems, such as those described in International PCT application Nos.WO 95/12808; WO 96/01836 and WO 96/07917 and also in arrays, such asthose described in U.S. Pat. No. 5,451,683, which are each hereinincorporated by reference. The self-addressable chips are such that eachindividual well may be addressed one at a time in the presence of therest by changing the charge at a single microlocation and then sendingthe analytes or reagents via free flow electrophoresis throughout, butassembly occurs only at that location after the chip has been assembled.These devices are modified for use in the methods herein by replacingthe disclosed detection means with the luciferase/luciferin systems.

In another embodiment provided herein, electrodes, an anode and cathode,are located at the bottom and top of each well, respectively, to allowfor the delivery of analytes and reagents by free flow electrophoresis.The antibodies are attached to each location on a MYLAR (orientedpolyethylene terephthalate) layer prior to assembling the chip (using,for example, a dot matrix printer). Thus, it is nonself-addressable inthat is has a plurality of individual wells each containing a photodiodeincorporated into the semiconductor layer at the bottom of each well.

In practice, for example, specific anti-ligands, e.g., antibodies, maybe attached directly to the matrix of the chip or to a middle reflectivesupport matrix, such as heat stable MYLAR, positioned in the center ofeach well. The sample is contacted with the chip, washed and a pluralityof secondary antibody-luciferase conjugates or protein fusions areadded. The wells are washed and the remaining components of thebioluminescent reaction are added to initiate the reaction. Lightproduced in a well is detected by the photodiode, photomultiplier, CCD(charge coupled device) or other suitable detector in the semiconductorlayer and the signal is relayed to a processing unit, typically acomputer. The processing unit displays the well or wells that arepositive. Each well corresponds to a particular ligand, therebypermitting identification of the infectious agents. All steps may beautomated.

The design, fabrication, and uses of nonself-addressable andprogrammable, self-addressable and self-assembling microelectronicsystems and devices which actively carry out controlled multi-step andmultiplex reactions in microscopic formats for detecting theelectromagnetic emissions of a bioluminescent reaction are providedherein. The reactions include, but are not limited to, most molecularbiological procedures, such as nucleic acid and protein nucleic acidhybridizations, antibody/antigen reactions, and related clinicaldiagnostics.

The resulting chips, which includes a silicon matrix and photodiodes orother light detecting means, are provided. The silicon may be depositedusing enzymatic deposition, similar to the enzymatic deposition byradiolarains and diatoms. Also provided are chips in which theabsorption of silica or derivatives thereof is advantageously employedas a detection means. Such silica has an absorption maxima at about 705nm, which is the wavelength emitted by Aristostomias bioluminescencegenerating system. Enzymatic methods for depositing silicon on thesurface of a matrix are also provided herein.

Also provided herein is a synthetic synapse. A suitable enzyme,particularly, acetylcholine esterase is fused to a luciferase, such asby recombinant expression. The luciferase is either in an inactive oractive conformation. Suitable mutations in either protein may beselected to insure that luciferase can undergo appropriateconformational changes as described herein. The resulting fusion isattached to a chip, such as a chip provided herein. Upon binding of theligand to the enzyme, such as the binding of acetylcholine to theesterase, the linked luciferase is, if previously inactive, is activatedby the binding, or if previously active, is inactivated by the binding.In the presence of the remaining components of a bioluminescencegenerating system, light is produced (or is quenched), which change isdetected by the photodiodes associated with the chip. This detectiongenerates an signal that is processed, such as by a computer, and istransmitted by appropriate means, such as fiber, to an electrode, whichis attached to any desired device or effector, particularly a muscle.Upon receipt of the signal, work, such as a muscle twitch, occurs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram of a microelectronic device fordetecting and identifying analytes in a biological sample usingbioluminescence, the microelectronic device including an array ofmicro-locations and a photodetector optically coupled to eachmicro-location for detecting the bioluminescence emitted at thecorresponding micro-location;

FIG. 2 is a top view of the die for the microelectronic device of FIG. 1showing the photodetector array disposed on a semiconductor substrate;

FIG. 3 is a perspective view of the microelectronic device of FIG. 1including the die of FIG. 2 housed in a ceramic dual in-line package(DIP), and FIG. 3A is a magnified view showing the test well formed inthe DIP in detail;

FIG. 4 is a schematic diagram showing a pixel unit cell circuit fordetecting the bioluminescence emitted at each micro-location in thearray;

FIG. 5 is a graph showing the voltage levels at three nodes of the pixelunit cell circuit of FIG. 4 as a function of time during operation ofthe device;

FIG. 6 is a system block diagram showing the microelectronics device ofFIG. 1 mounted on an adaptor circuit board and serially interfaced to acomputer programmed to read the serial output data stream, to correlatethe output data with the array of micro-locations, to integrate the datacorrelated with each micro-location for a predetermined time period setusing an input device, to identify the analytes present in thebiological medium by reference to an analyte map, and to display theresults on an output device; and

FIG. 7 shows the microelectronics device of FIG. 1 received on a circuitboard which does not require the user to directly handle the package.

FIG. 8 is a schematic cross-sectional diagram of a three layermulti-well CCD chip (a chip containing a photodiode/CCD).

FIG. 9 shows a blown-up schematic diagram of a multi-well CCD chipbottom layer and middle reflective layer and schematic diagram of anindividual well.

FIG. 10 shows a blown-up schematic diagram of specific antibodiesattached to the middle reflective layer of the multi-well CCD chip ofFIG. 8.

FIG. 11 is a cross-section of an individual well indicating the relativepositions of the CCD, reflective mirror layer and the cathode and anode.Antibodies attached to the middle reflective layer hang inverted abovethe photodiode. Bound antigen is detected using an antibody-luciferasefusion protein, and light generated from the bioluminescent reaction isdetected by the photodiode and relayed to a processing unit foridentification.

FIG. 12 is the cross-section of three self-addressable micro-locationsfabricated using microlithographic techniques [see, International PCTapplication No. WO 96/01836]. Included are arrows denoting thepositioning of photodiodes.

FIG. 13 is the cross-section of a microlithographically fabricatedmicro-location; antibodies or other receptors are linked to theattachment layer.

FIG. 14 is a schematic representation of a self-addressable 64micro-location chip which was actually fabricated, addressed witholigonucleotides, and tested.

FIG. 15 shows a blown-up schematic diagram of a micro-machined 96micro-locations device.

FIG. 16 is the cross-section of a micro-machined device.

FIG. 17 shows a schematic representation of an artificialsilicon-synapse.

FIG. 18 shows a detailed schematic view of an acetylcholineesterase-luciferase fusion protein and an acetylcholineesterase-fluorochrome conjugate used in the silicon-synapse.

FIG. 19 depicts the methodology for the placement of silicon-synapsesand electrodes in the human spinal cord to bypass a permenant spinalcord lesion.

FIG. 20 depicts a scheme for operation of chips described herein indiagnostic assays for detecting infectious microorganisms.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS TABLE OF CONTENTS

A. Definitions

B. Bioluminescence generating systems

1. General description

a. Luciferases

b. Luciferins

c. Activators

d. Reactions

2. Ctenophore and coelenterate systems

a. The aequorin system

(1) Aequorin photoprotein

(2) Luciferin

b. The Renilla system

3. Crustacean, particular Cyrpidina [Vargula], systems

a. Vargula luciferase

(1) Purification from Cypridina

(2) Preparation by Recombinant Methods

b. Vargula luciferin

c. Reaction

4. Insect bioluminescence generating systems including fireflies, clickbeetles, and other insect systems

a. Luciferase

b. Luciferin

c. Reaction

5. Bacterial systems

a. Luciferases

b. Luciferins

c. Reactions

6. Other systems

a. Dinoflagellate bioluminescence generating systems

b. Systems from molluscs, such as Latia and Pholas

c. Earthworms and other annelids

d. Glow worms

e. Marine polycheate worm systems

f. South American railway beetle

7. Fluorescent proteins

a. Green and blue fluorescent proteins

b. Phycobiliproteins

C. Design and Fabrication of Chips

1. Nonself-addressable chips

2. Self-addressable chips

a. Matrix materials

b. Fabrication procedures

i. Microlithography

ii. Micromachining

c. Self addressing of chips

3. Attachment of biological molecules to chips

a. Derivatization of silica substrates

b. Attachment of biological molecules

D. Formation of luciferase conjugates

1. Linkers

2. Luciferase fusion proteins

3. Nucleic acid and peptide nucleic acid conjugates

E. Radiolarians and diatoms for deposition of silicon on matrices

F. Methods employing the chip

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference herein.

As used herein, chemiluminescence refers to a chemical reaction in whichenergy is specifically channeled to a molecule causing it to becomeelectronically excited and subsequently to release a photon therebyemitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy. Bioluminescence refers to the subsetof chemiluminescence reactions that involve luciferins and luciferases(or the photoproteins). Bioluminescence does not herein includephosphorescence.

As used herein, bioluminescence, which is a type of chemi-luminescence,refers to the emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin. Bioluminescence isgenerated by an enzyme or other protein [luciferase] that is anoxygenase that acts on a substrate luciferin [a bioluminescencesubstrate] in the presence of molecular oxygen and transforms thesubstrate to an excited state, which upon return to a lower energy levelreleases the energy in the form of light.

As used herein, the substrates and enzymes for producing bioluminescenceare generically referred to as luciferin and luciferase, respectively.When reference is made to a particular species thereof, for clarity,each generic term is used with the name of the organism from which itderives, for example, bacterial luciferin or firefly luciferase.

As used herein, luciferase refers to oxygenases that catalyze a lightemitting reaction. For instance, bacterial luciferases catalyze theoxidation of flavin mononucleotide [FMN] and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina [Vargula]luciferin, and another class of luciferases catalyzes the oxidation ofColeoptera luciferin.

Thus, luciferase refers to an enzyme or photoprotein that catalyzes abioluminescent reaction [a reaction that produces bioluminescence]. Theluciferases, such as firefly and Renilla luciferases, that are enzymeswhich act catalytically and are unchanged during the bioluminescencegenerating reaction. The luciferase photoproteins, such as the aequorinand obelin photoproteins to which luciferin is non-covalently bound, arechanged, such as by release of the luciferin, during bioluminescencegenerating reaction. The luciferase is a protein that occurs naturallyin an organism or a variant or mutant thereof, such as a variantproduced by mutagenesis that has one or more properties, such as thermalor pH stability, that differ from the naturally-occurring protein.Luciferases and modified mutant or variant forms thereof are well known.

Thus, reference, for example, to “Renilla luciferase” means an enzymeisolated from member of the genus Renilla or an equivalent moleculeobtained from any other source, such as from another Anthozoa, or thathas been prepared synthetically.

The luciferases and luciferin and activators thereof are referred to asbioluminescence generating reagents or components. Typically, a subsetof these reagents will be provided during the assay or otherwiseimmobilized at particular locations on the surface of the chip.Bioluminescence will be produced upon contacting the chip surface withthe remaining reagents and the light produced is detected by thephotodiodes at those locations of the array where a specific target hasbeen detected by the immobilized anti ligand. Thus, as used herein, thecomponent luciferases, luciferins, and other factors, such as O₂, Mg²⁺,Ca²⁺ are also referred to as bioluminescence generating reagents [oragents or components].

As used herein, “not strictly catalytically” means that the photoproteinacts as a catalyst to promote the oxidation of the substrate, but it ischanged in the reaction, since the bound substrate is oxidized and boundmolecular oxygen is used in the reaction. Such photoproteins areregenerated by addition of the substrate and molecular oxygen underappropriate conditions known to those of skill in this art.

As used herein, bioluminescence substrate refers to the compound that isoxidized in the presence of a luciferase, and any necessary activators,and generates light. These substrates are referred to as luciferins,which are substrates that undergo oxidation in a bioluminescencereaction. These bioluminescence substrates include any luciferin oranalog thereof or any synthetic compound with which a luciferaseinteracts to generate light. Preferred substrates are those that areoxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina [alsoknown as Vargula] luciferin [coelenterazine], bacterial luciferin, aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction the producesbioluminescence.

As used herein, capable of conversion into a bioluminescence substratemeans susceptible to chemical reaction, such as oxidation or reduction,that yields a bioluminescence substrate. For example, the luminescenceproducing reaction of bioluminescent bacteria involves the reduction ofa flavin mononucleotide group (FMN) to reduced flavin mononucleotide(FMNH₂) by a flavin reductase enzyme. The reduced flavin mononucleotide[substrate] then reacts with oxygen [an activator] and bacterialluciferase to form an intermediate peroxy flavin that undergoes furtherreaction, in the presence of a long-chain aldehyde, to generate light.With respect to this reaction, the reduced flavin and the long chainaldehyde are substrates.

As used herein, bioluminescence system [or bioluminescence generatingsystem] refers to the set of reagents required for abioluminescence-producing reaction. Thus, the particular luciferase,luciferin and other substrates, solvents and other reagents that may berequired to complete a bioluminescent reaction form a bioluminescencesystem. Therefore, a bioluminescence system (or equivalently abioluminescence generating system) refers to any set of reagents that,under appropriate reaction conditions, yield bioluminescence.Appropriate reaction conditions refers to the conditions necessary for abioluminescence reaction to occur, such as pH, salt concentrations andtemperature. In general, bioluminescence systems include abioluminescence substrate (a luciferin), a luciferase, which includesenzymes luciferases and photoproteins, and one or more activators. Aparticular bioluminescence system may be identified by reference to thespecific organism from which the luciferase derives; for example, theVargula [also called Cypridina] bioluminescence system (or Vargulasystem) includes a Vargula luciferase, such as a luciferase isolatedfrom the ostracod, Vargula or produced using recombinant means ormodifications of these luciferases. This system would also include theparticular activators necessary to complete the bioluminescencereaction, such as oxygen and a substrate with which the luciferasereacts in the presence of the oxygen to produce light.

As used herein, ATP, AMP, NAD+ and NADH refer to adenosine triphosphate,adenosine monophosphate, nicotinamide adenine dinucleotide (oxidizedform) and nicotinamide adenine dinucleotide (reduced form),respectively.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When “equivalent” is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with onlyconservative amino acid substitutions [see, e.g., Table 2, below] thatdo not substantially alter the activity or function of the protein orpeptide. When “equivalent” refers to a property, the property does notneed to be present to the same extent [e.g., two peptides can exhibitdifferent rates of the same type of enzymatic activity], but theactivities are preferably substantially the same. “Complementary,” whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, preferably with less than 25%,more preferably with less than 15%, even more preferably with less than5%, most preferably with no mismatches between opposed nucleotides.Preferably the two molecules will hybridize under conditions of highstringency.

As used herein: stringency of hybridization in determining percentagemismatch is as follows:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C. It is understood thatequivalent stringencies may be achieved using alternative buffers, saltsand temperatures.

As used herein, peptide nucleic acid refers to nucleic acid analogs inwhich the ribose-phosphate backbone is replaced by a backbone heldtogether by amide bonds.

The term “substantially” varies with the context as understood by thoseskilled in the relevant art and generally means at least 70%, preferablymeans at least 80%, more preferably at least 90%, and most preferably atleast 95%.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon administration ofa compound, composition or other mixture. Biological activities may beobserved in in vitro systems designed to test or use such activities.Thus, for purposes herein the biological activity of a luciferase is itsoxygenase activity whereby, upon oxidation of a substrate, light isproduced.

As used herein, a composition refers to a any mixture. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a combination refers to any association between two oramong more items.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, macromolecules are intended to generically encompass allmolecules that would be linked to a solid support for diagnostic assays.The macromolecules include, but are not limited to: proteins, organicmolecules, nucleics acids, viruses, viral capsids, phage, cells ormembranes thereof or portions of viruses, viral capsids, phage, cells ormembranes. Of particular interest herein, are macromolecules thatspecifically bind to an analyte of interest. Analytes of interest arethose present in body fluids and other biological samples.

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors may be naturally-occurring or syntheticmolecules. Receptors may also be referred to in the art as anti-ligands.As used herein, the receptor and anti-ligand are interchangeable.Receptors can be used in their unaltered state or as aggregates withother species. Receptors may be attached, covalently or noncovalently,or in physical contact with, to a binding member, either directly orindirectly via a specific binding substance or linker. Examples ofreceptors, include, but are not limited to: antibodies, cell membranereceptors surface receptors and internalizing receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants[such as on viruses, cells, or other materials], drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to:

a) enzymes: specific transport proteins or enzymes essential to survivalof microorganisms, which could serve as targets for antibiotic [ligand]selection;

b) antibodies: identification of a ligand-binding site on the antibodymolecule that combines with the epitope of an antigen of interest may beinvestigated; determination of a sequence that mimics an antigenicepitope may lead to the development of vaccines of which the immunogenis based on one or more of such sequences or lead to the development ofrelated diagnostic agents or compounds useful in therapeutic treatmentssuch as for auto-immune diseases

c) nucleic acids: identification of ligand, such as protein or RNA,binding sites;

d) catalytic polypeptides: polymers, preferably polypeptides, that arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products; such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,in which the functionality is capable of chemically modifying the boundreactant [see, e.g., U.S. Pat. No. 5,215,899];

e) hormone receptors: determination of the ligands that bind with highaffinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

f) opiate receptors: determination of ligands that bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, complementary refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

As used herein, a ligand-receptor pair or complex formed when twomacromolecules have combined through molecular recognition to form acomplex.

As used herein, an epitope refers to a portion of an antigen moleculethat is delineated by the area of interaction with the subclass ofreceptors known as antibodies.

As used herein, a ligand is a molecule that is specifically recognizedby a particular receptor. Examples of ligands, include, but are notlimited to, agonists and antagonists for cell membrane receptors, toxinsand venoms, viral epitopes, hormones [e.g., steroids], hormonereceptors, opiates, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

As used herein, an anti-ligand (AL.sub.i): An anti-ligand is a moleculethat has a known or unknown affinity for a given ligand and can beimmobilized on a predefined region of the surface. Anti-ligands may benaturally-occurring or manmade molecules. Also, they can be employed intheir unaltered state or as aggregates with other species. Anti-ligandsmay be reversibly attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. By“reversibly attached” is meant that the binding of the anti-ligand (orspecific binding member or ligand) is reversible and has, therefore, asubstantially non-zero reverse, or unbinding, rate. Such reversibleattachments can arise from noncovalent interactions, such aselectrostatic forces, van der Waals forces, hydrophobic (i.e., entropic)forces, and the like. Furthermore, reversible attachments also may arisefrom certain, but not all covalent bonding reactions. Examples include,but are not limited to, attachment by the formation of hemiacetals,hemiketals, imines, acetals, ketals, and the like (See, Morrison et al.,“Organic Chemistry”, 2nd ed., ch. 19 (1966), which is incorporatedherein by reference). Examples of anti-ligands which can be employed inthe methods and devices herein include, but are not limited to, cellmembrane receptors, monoclonal antibodies and antisera reactive withspecific antigenic determinants (such as on viruses, cells or othermaterials), hormones, drugs, oligonucleotides, peptides, peptide nucleicacids, enzymes, substrates, cofactors, lectins, sugars,oligosaccharides, cells, cellular membranes, and organelles.

As used herein, a substrate refers to any matrix that is used eitherdirectly or following suitable derivatization, as a solid support forchemical synthesis, assays and other such processes. Preferredsubstrates herein, are silicon substrates or siliconized substrates thatare derivatized on the surface intended for linkage of anti-ligands andligands and other macromolecules, including the fluorescent proteins,phycobiliproteins and other emission shifters.

As used herein, a matrix refers to any solid or semisolid or insolublesupport on which the molecule of interest, typically a biologicalmolecule, macromolecule, organic molecule or biospecific ligand islinked or contacted. Typically a matrix is a substrate material having arigid or semi-rigid surface. In many embodiments, at least one surfaceof the substrate will be substantially flat, although in someembodiments it may be desirable to physically separate synthesis regionsfor different polymers with, for example, wells, raised regions, etchedtrenches, or other such topology. Matrix materials include any materialsthat are used as affinity matrices or supports for chemical andbiological molecule syntheses and analyses, such as, but are not limitedto: polystyrene, polycarbonate, polypropylene, nylon, glass, dextran,chitin, sand, pumice, polytetrafluoroethylene, agarose, polysaccharides,dendrimers, buckyballs, polyacrylamide, Kieselguhr-polyacrlamidenon-covalent composite, polystyrene-polyacrylamide covalent composite,polystyrene-PEG [polyethyleneglycol] composite, silicon, rubber, andother materials used as supports for solid phase syntheses, affinityseparations and purifications, hybridization reactions, immunoassays andother such applications.

As used herein, the attachment layer refers the surface of the chipdevice to which molecules are linked. Typically, the chip is asemiconductor device, which is coated on a least a portion of thesurface to render it suitable for linking molecules and inert to anyreactions to which the device is exposed. Molecules are linked eitherdirectly or indirectly to the surface, linkage may be effected byabsorption or adsorption, through covalent bonds, ionic interactions orany other interaction. Where necessary the attachment layer is adapted,such as by derivatization for linking the molecules.

B. Bioluminescence Generating Systems

A bioluminescence generating system refers to the components that arenecessary and sufficient to generate bioluminescence. These include aluciferase, luciferin and any necessary co-factors or conditions.Virtually any bioluminescence generating system known to those of skillin the art will be amenable to use in the apparatus, systems,combinations and methods provided herein. Factors for consideration inselecting a bioluminescence generating system, include, but are notlimited to: the desired assay and biological fluid used in combinationwith the bioluminescence; the medium in which the reaction is run;stability of the components, such as temperature or pH sensitivity;shelf life of the components; sustainablity of the light emission,whether constant or intermittent; availability of components; desiredlight intensity; and other such factors.

1. General Description

In general, bioluminescence refers to an energy-yielding chemicalreaction in which a specific chemical substrate, a luciferin, undergoesoxidation, catalyzed by an enzyme, a luciferase. Bioluminescentreactions are easily maintained, requiring only replenishment ofexhausted luciferin or other substrate or cofactor or other protein, inorder to continue or revive the reaction. Bioluminescence generatingreactions are well known to those of skill in this art and any suchreaction may be adapted for use in combination with apparatus, systemsand methods described herein.

There are numerous organisms and sources of bioluminescence generatingsystems, and some representative genera and species that exhibitbioluminescence are set forth in the following table [reproduced in partfrom Hastings in (1995) Cell Physiology:Source Book, N. Sperelakis(ed.), Academic Press, pp 665-681]:

TABLE 1 Representative luminous organism Type of Organism Representativegenera Bacteria Photobacterium Vibrio Xenorhabdus Mushrooms Panus,Armillaria Pleurotus Dinoflagellates Gonyaulax Pyrocystis NoctilucaCnidaria (coelenterates) Jellyfish Aequorea Hydroid Obelia Sea PansyRenilla Ctenophores Mnemiopsis Beroe Annelids Earthworms DiplocardiaMarine polychaetes Chaetopterus, Phyxotrix Syllid fireworm OdontosyllisMolluscs Limpet Latia Clam Pholas Squid Heteroteuthis HeterocarpusCrustacea Ostracod Vargula (Cypridina) Shrimp (euphausids)Meganyctiphanes Acanthophyra Oplophorus Gnathophausia Decapod SergestesCopepods Insects Coleopterids (beetles) Firefly Photinus, Photuris Clickbeetles Pyrophorus Railroad worm Phengodes, Phrixothrix Diptera (flies)Arachnocampa Echinoderms Brittle stars Ophiopsila Sea cucumbersLaetmogone Chordates Tunicates Pyrosoma Fish Cartilaginous Squalus BonyPonyfish Leiognathus Flashlight fish Photoblepharon Angler fishCryptopsaras Midshipman Porichthys Lantern fish Benia Shiny loosejawAristostomias Hatchet fish Agyropelecus and other fish PachystomiasMalacosteus Midwater fish Cyclothone Neoscopelus Tarletonbeania

Other bioluminescent organisms contemplated for use as sources ofbioluminescence generating systems herein include, but are not limitedto, Gonadostomias, Gaussia, Halisturia, Vampire squid, Glyphus,Mycotophids (fish), Vinciguerria, Howella, Florenciella, Chaudiodus,Melanocostus, Paracanthus, Atolla, Pelagia, Pitilocarpus, Acanthophyra,Siphonophore, Periphylla and Sea Pens (Stylata).

It is understood that a bioluminescence generating system may beisolated from natural sources, such as those in the above Table, or maybe produced synthetically. In addition, for uses herein, the componentsneed only be sufficiently pure so that mixture thereof, underappropriate reaction conditions, produces a glow. Thus it has beenfound, in some embodiments, a crude extract or merely grinding up theorganism may be adequate. Generally, however, substantially purecomponents are used, but, where necessary, the precise purity can bedetermined empirically. Also, components may be synthetic componentsthat are not isolated from natural sources. DNA encoding luciferases isavailable [see, e.g., SEQ ID Nos. 1-13] and has been modified [see,e.g., SEQ ID Nos. 3 and 10-13] and synthetic and alternative substrateshave been devised. The DNA listed herein is only representative of theDNA encoding luciferases that is available.

Any bioluminescence generating system, whether synthetic or isolatedform natural sources, such as those set forth in Table 1, elsewhereherein or known to those of skill in the art, is intended for use in thechip devices, combinations, systems and methods provided herein.Chemiluminescence systems per se, which do not rely on oxygenases[luciferases] are not encompassed herein.

a. Luciferases

Luciferases refer to any compound that, in the presence of any necessaryactivators, catalyze the oxidation of a bioluminescence substrate[luciferin] in the presence of molecular oxygen, whether free or bound,from a lower energy state to a higher energy state such that thesubstrate, upon return to the lower energy state, emits light. Forpurposes herein, luciferase is broadly used to encompass enzymes thatact catalytically to generate light by oxidation of a substrate and alsophotoproteins, such as aequorin, that act, though not strictlycatalytically [since such proteins are exhausted in the reaction], inconjunction with a substrate in the presence of oxygen to generatelight. These luciferases, including photoproteins, such as aequorin, areherein also included among the luciferases. These reagents include thenaturally-occurring luciferases [including photoproteins], proteinsproduced by recombinant DNA, and mutated or modified variants thereofthat retain the ability to generate light in the presence of anappropriate substrate, co-factors and activators or any other suchprotein that acts as a catalyst to oxidize a substrate, whereby light isproduced.

Generically, the protein that catalyzes or initiates the bioluminescentreaction is referred to as a luciferase, and the oxidizable substrate isreferred to as a luciferin. The oxidized reaction product is termedoxyluciferin, and certain luciferin precursors are termed etioluciferin.Thus, for purposes herein bioluminescence encompasses light produced byreactions that are catalyzed by [in the case of luciferases that actenzymatically] or initiated by [in the case of the photoproteins, suchas aequorin, that are not regenerated in the reaction] a biologicalprotein or analog, derivative or mutant thereof.

For clarity herein, these catalytic proteins are referred to asluciferases and include enzymes such as the luciferases that catalyzethe oxidation of luciferin, emitting light and releasing oxyluciferin.Also included among luciferases are photoproteins, which catalyze theoxidation of luciferin to emit light but are changed in the reaction andmust be reconstituted to be used again. The luciferases may be naturallyoccurring or may be modified, such as by genetic engineering to improveor alter certain properties. As long as the resulting molecule retainsthe ability to catalyze the bioluminescent reaction, it is encompassedherein.

Any protein that has luciferase activity [a protein that catalyzesoxidation of a substrate in the presence of molecular oxygen to producelight as defined herein] may be used herein. The preferred luciferasesare those that are described herein or that have minor sequencevariations. Such minor sequence variations include, but are not limitedto, minor allelic or species variations and insertions or deletions ofresidues, particularly cysteine residues. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. MolecularBiologyofthe Gene, 4th Edition, 1987, TheBenjamin/Cummings Pub. co., p.224). Such substitutions are preferablymade in accordance with those set forth in TABLE 2 as follows:

TABLE 2 Original residue Conservative substitution Ala (A) Gly; Ser Arg(R) Lys Asn (N) Gln; His Cys (C) Ser; neutral amino acid Gln (Q) Asn Glu(E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile;Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; TyrSer (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions. Any suchmodification of the polypeptide may be effected by any means known tothose of skill in this art.

The luciferases may be obtained commercially, isolated from naturalsources, expressed in host cells using DNA encoding the luciferase, orobtained in any manner known to those of skill in the art. For purposesherein, crude extracts obtained by grinding up selected source organismsmay suffice. Since large quantities of the luciferase may be desired,isolation of the luciferase from host cells is preferred. DNA for suchpurposes is widely available as are modified forms thereof.

Examples of luciferases include, but are not limited to, those isolatedfrom the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),those isolated from the coelenterates Aequorea (aequorin), Obelia(obelin), Pelagia, the Renilla luciferase, the luciferases isolated fromthe mollusca Pholas (pholasin), the luciferases isolated from theAristostomias and Porichthys fish and from the ostracods, such asCypridina (also referred to as Vargula). Preferred luciferases for useherein are the Aequorin protein, Renilla luciferase and Cypridina [alsocalled Vargula] luciferase [see, e.g., SEQ ID Nos. 1, 2, and 4-13].Also, preferred are luciferases which react to produce red and/or nearinfrared light. These include luciferases found in species ofAristostomias, such as A. scintillans, Pachystomias, Malacosteus, suchas M. niger.

b. Luciferins

The substrates for the reaction include any molecule(s) with which theluciferase reacts to produce light. Such molecules include thenaturally-occurring substrates, modified forms thereof, and syntheticsubstrates [see, e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828].Exemplary luciferins include those described herein, as well asderivatives thereof, analogs thereof, synthetic substrates, such asdioxetanes [see, e.g., U.S. Pat. Nos. 5,004,565 and 5,455,357], andother compounds that are oxidized by a luciferase in a light-producingreaction [see, e.g., U.S. Pat. Nos. 5,374,534, 5,098,828 and 4,950,588].Such substrates also may be identified empirically by selectingcompounds that are oxidized in bioluminescent reactions.

c. Activators

The bioluminescence generating systems also require additionalcomponents discussed herein and known to those of skill in the art. Allbioluminescent reactions require molecular oxygen in the form ofdissolved or bound oxygen. Thus, molecular oxygen, dissolved in water orin air or bound to a photoprotein, is the activator for bioluminescencereactions. Depending upon the form of the components, other activatorsinclude, but are not limited to, ATP [for firefly luciferase], flavinreductase [bacterial systems] for regenerating FMNH₂ from FMN, and Ca²⁺or other suitable metal ion [aequorin].

Most of the systems provided herein will generate light when theluciferase and luciferin are mixed and exposed to air or water. Thesystems that use photoproteins that have bound oxygen, such as aequorin,however, will require exposure to Ca²⁺ [or other suitable metal ion],which can be provided in the form of an aqueous composition of a calciumsalt. In these instances, addition of a Ca²⁺ [or other suitable metalion] to a mixture of luciferase [aequorin] and luciferin [such ascoelenterazine] will result in generation of light. The Renilla systemand other Anthozoa systems also require Ca²⁺ [or other suitable metalion].

If crude preparations are used, such as ground up Cypridina [shrimp] orground fireflies, it may be necessary to add only water. In instances inwhich fireflies [or a firefly or beetle luciferase] are used thereaction may only require addition ATP. The precise components will beapparent, in light of the disclosure herein, to those of skill in thisart or may be readily determined empirically.

It is also understood that these mixtures will also contain anyadditional salts or buffers or ions that are necessary for each reactionto proceed. Since these reactions are well-characterized, those of skillin the art will be able to determine precise proportions and requisitecomponents. Selection of components will depend upon the chip device andsystem, the assay to be preformed and the luciferase. Variousembodiments are described and exemplified herein; in view of suchdescription, other embodiments will be apparent.

d. Reactions

In all embodiments, up to all but one component of a bioluminescencegenerating system will be bound directly or indirectly to theappropriate locations of the chip or otherwise immobilized at thosepositions of the array in which the presence of analyte, preferably aninfectious agent, is detected. When bioluminescence is desired, theremaining component(s) will be added to the surface of the chip and thelight produced at those locations of the array is detected by thephotodiodes of the chip.

In general, since the result to be achieved is the production of lightthat can be detected by the photodiodes of the chip or visible to thenaked eye for the purposes herein, the precise proportions and amountsof components of the bioluminescence reaction need not be stringentlydetermined or met. They must be sufficient to produce light. Generally,an amount of luciferin and luciferase sufficient to generate a readilydetectable signal or a visible glow is used; this amount can be readilydetermined empirically and is dependent upon the selected system andselected application.

For purposes herein, such amount is preferably at least theconcentrations and proportions used for analytical purposes by those ofskill in the such arts. Higher concentrations or longer integrationtimes may be used if the glow is not sufficiently bright to be detectedby photodiodes in the chip. Also because the conditions in which thereactions are used are not laboratory conditions and the components aresubject to storage, higher concentration may be used to overcome anyloss of activity. Typically, the amounts are 1 mg, preferably 10 mg andmore preferably 100 mg, of a luciferase per liter of reaction mixture or1 mg, preferably 10 mg, more preferably 100 mg. Such luciferases may beproduced by drying a composition containing at least about 0.01 mg/l,and typically 0.1 mg/l, 1 mg/l, 10 mg/l or more of each component. Theamount of luciferin is also between about 0.01 and 100 mg/l, preferablybetween 0.1 and 10 mg/l, additional luciferin can be added to many ofthe reactions to continue the reaction. In embodiments in which theluciferase acts catalytically and does not need to be regenerated, loweramounts of luciferase can be used. In those in which it is changedduring the reaction, it also can be replenished; typically higherconcentrations will be selected. Ranges of concentration per liter [orthe amount of coating on substrate the results from contacting with suchcomposition] of each component on the order of 0.1 to 20 mg, preferably0.1 to 10 mg, more preferably between about 1 and 10 mg of eachcomponent will be sufficient. When preparing coated substrates, asdescribed herein, greater amounts of coating compositions containinghigher concentrations of the luciferase or luciferin may be used.

Thus, for example, in presence of calcium, 5 mg of luciferin, such ascoelenterazine, in one liter of water will glow brightly for at leastabout 10 to 20 minutes, depending on the temperature of the water, whenabout 10 mgs of luciferase, such as aequorin photoprotein luciferase orluciferase from Renilla, is added thereto. Increasing the concentrationof luciferase, for example, to 100 mg/l, provides a particularlybrilliant display of light.

If desired, the onset of the bioluminescent reaction can be delayed byadding an, an inhibitor, for example magnesium, of the bioluminescencegenerating reaction. Also, where inhibition is not desired, theconcentration of free magnesium may be reduced by addition of asufficient amount of chelating agent, such as ethylenediaminetetraaceticacid [EDTA]. The amount of EDTA and also calcium can be empiricallydetermined to appropriately chelate magnesium, without inhibiting orpreventing the desired bioluminescence.

It is understood, that concentrations and amounts to be used depend uponthe selected luciferase, the desired bacterial target, the concentrationand amount of light absorbed by the immobilized anti ligand, the size ofthe photodiode array and these may be readily determined empirically.Proportions, particularly those used when commencing an empiricaldetermination, are generally those used for analytical purposes, andamounts or concentrations are at least those used for analyticalpurposes, but the amounts can be increased, particularly if a sustainedand brighter glow is desired.

2. Ctenophore and Coelenterate Systems

Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),and coelenterates, such as Aequorea (aequorin), Obelia (obelin) andPelagia, produce bioluminescent light using similar chemistries [see,e.g., Stephenson et al. (1981) Biochimica et Biophysica Acta 678:65-75;Hart et al. (1979) Biochemistry 18:2204-2210; International PCTApplication No. WO94/18342, which is based on U.S. application Ser. No.08/017,116, U.S. Pat. No. 5,486,455 and other references and patentscited herein]. The Aequorin and Renilla systems are representative andare described in detail herein as exemplary and as among the presentlypreferred systems. The Aequorin and Renilla systems can use the sameluciferin and produce light using the same chemistry, but eachluciferase is different. The Aequorin luciferase aequorin, as well as,for example, the luciferases mnemiopsin and berovin, is a photoproteinthat includes bound oxygen and bound luciferin, requires Ca²⁺ [or othersuitable metal ion] to trigger the reaction, and must be regenerated forrepeated use; whereas, the Renilla luciferase acts as a true enzymebecause it is unchanged during the reaction and it requires dissolvedmolecular oxygen.

a. The Aequorin System

The aequorin system is well known [see, e.g., Tsuji et al. (1986)“Site-specific mutagenesis of the calcium-binding photoproteinaequorin,” Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al.(1985) “Cloning and Expression of the cDNA Coding for Aequorin, aBioluminescent Calcium-Binding Protein,” Biochemical and BiophysicalResearch Communications 126:1259-1268; Prasher et al. (1986) Methods inEnzymology 133:288-297; Prasher, et al. (1987) “Sequence Comparisons ofcDNAs Encoding for Aequorin Isotypes,” Biochemistry 26:1326-1332;Charbonneau et al. (1985) “Amino Acid Sequence of the Calcium-DependentPhotoprotein Aequorin,” Biochemistry 24:6762-6771; Shimomura et al.(1981) “Resistivity to denaturation of the apoprotein of aequorin andreconstitution of the luminescent photoprotein from the partiallydenatured apoprotein,” Biochem. J. 199:825-828; Inouye et al. (1989) J.Biochem. 105:473-477; Inouye et al. (1986) “Expression of ApoaequorinComplementary DNA in Escherichia coli,” Biochemistry 25:8425-8429;Inouye et al. (1985) “Cloning and sequence analysis of cDNA for theluminescent protein aequorin,” Proc. Natl. Acad. Sci. USA 82:3154-3158;Prendergast, et al. (1978) “Chemical and Physical Properties of Aequorinand the Green Fluorescent Protein Isolated from Aequorea forskalea” J.Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 064 A1;European Patent Application 0 226 979 A2, European Patent Application 0245 093 A1 and European Patent Specification 0 245 093 B1; U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form,described in U.S. Pat. No. 5,162,227, European Patent Application 0 540064 A1 and Sealite Sciences Technical Report No. 3 (1994), iscommercially available from Sealite, Sciences, Bogart, Ga. asAQUALITE®].

This system is among the preferred systems for use herein. As will beevident, since the aequorin photoprotein includes noncovalently boundluciferin and molecular oxygen, it is suitable for storage in this formas a lyophilized powder or encapsulated into a selected deliveryvehicle. The system can be encapsulated into pellets, such as liposomesor other delivery vehicles, or stored in single chamber dual or othermultiple chamber ampules. When used, the photoproteins will beconjugated to an anti ligand, bound to the specified positions in thearray and contacted with a composition, even tap water, that containsCa²⁺ [or other suitable metal ion], to produce a mixture that glows atthat particular location of the array. The light is detected by thephotodiodes in the chip and the data signals are analyzed by theassociated computer processor. This system is preferred for use innumerous embodiments herein.

(1) Aequorin and Related Photoproteins

The photoprotein, aequorin, isolated from the jellyfish, Aequorea, emitslight upon the addition of Ca²⁺ [or other suitable metal ion]. Theaequorin photoprotein, which includes bound luciferin and bound oxygenthat is released by Ca²⁺, does not require dissolved oxygen.Luminescence is triggered by calcium, which releases oxygen and theluciferin substrate producing apoaqueorin.

The bioluminescence photoprotein aequorin is isolated from a number ofspecies of the jellyfish Aequorea. It is a 22 kilodalton [kD] molecularweight peptide complex [see, e.g., Shimomura et al. (1962) J. Cellularand Comp. Physiol. 59:233-238; Shimomura et al. (1969) Biochemistry8:3991-3997; Kohama et al. (1971) Biochemistry 10:4149-4152; andShimomura et al. (1972) Biochemistry 11:1602-1608]. The native proteincontains oxygen and a heterocyclic compound coelenterazine, a luciferin,[see, below] noncovalently bound thereto. The protein contains threecalcium binding sites. Upon addition of trace amounts Ca²⁺ [or othersuitable metal ion, such as strontium] to the photoprotein, it undergoesa conformational change the catalyzes the oxidation of the boundcoelenterazine using the protein-bound oxygen. Energy from thisoxidation is released as a flash of blue light, centered at 469 nm.Concentrations of calcium ions as low as 10⁻⁶ M are sufficient totrigger the oxidation reaction.

Naturally-occurring apoaequorin is not a single compound but rather is amixture of microheterogeneous molecular species. Aequoria jellyfishextracts contain as many as twelve distinct variants of the protein[see, e.g., Prasher et al. (187) Biochemistry 26:1326-1332; Blinks etal. (1975) Fed. Proc. 34:474]. DNA encoding numerous forms has beenisolated [see, e.g., SEQ ID Nos. 5-9 and 13].

The photoprotein can be reconstituted [see, e.g., U.S. Pat. No.5,023,181] by combining the apoprotein, such as a protein recombinantlyproduced in E. coli, with a coelenterazine, such as a syntheticcoelenterazine, in the presence of oxygen and a reducing agent [see,e.g., Shimomura et al. (1975) Nature 256:236-238; Shimomura et al.(1981) Biochemistry J. 199:825-828]), such as 2-mercaptoenthanol, andalso EDTA or EGTA [concentrations between about 5 to about 100 mM orhigher for applications herein] tie up any Ca²⁺ to prevent triggeringthe oxidation reaction until desired. DNA encoding a modified form ofthe apoprotein that does not require 2-mercaptoethanol forreconstitution is also available [see, e.g., U.S. Pat. No. 5,093,240].The reconstituted photoprotein is also commercially available [sold,e.g., under the trademark AQUALITE®, which is described in U.S. Pat. No.5,162,227].

The light reaction is triggered by adding Ca²⁺ at a concentrationsufficient to overcome the effects of the chelator and achieve the 10⁻⁶M concentration. Because such low concentrations of Ca²⁺ can trigger thereaction, for use in the methods herein, higher concentrations ofchelator may be included in the compositions of photoprotein.Accordingly, higher concentrations of added Ca²⁺ in the form of acalcium salt will be required. Precise amounts may be empiricallydetermined. For use herein, it may be sufficient to merely add water tothe photoprotein, which is provided in the form of a concentratedcomposition or in lyophilized or powdered form. Thus, for purposesherein, addition of small quantities of Ca²⁺ , such as those present inmost tap water or in phosphate buffered saline (PBS) or other suitablebuffers or possible in the moisture on the skin, should trigger thebioluminescence reaction.

Numerous isoforms of the aequorin apoprotein been identified isolated.DNA encoding these proteins has been cloned, and the proteins andmodified forms thereof have been produced using suitable host cells[see, e.g., U.S. Pat. Nos. 5,162,227, 5,360,728, 5,093,240; see, also,Prasher et al. (1985) Biophys. Biochem. Res. Commun. 126:1259-1268;Inouye et al. (1986) Biochemistry 25: 8425-84291]. U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,288,623; U.S. Pat. No. 5,422,266, U.S. Pat. No. 5,162,227 and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form iscommercially available form Sealite, Sciences, Bogart, Ga. asAQUALITE®]. DNA encoding apoaequorin or variants thereof is useful forrecombinant production of high quantities of the apoprotein. Thephotoprotein is reconstituted upon addition of the luciferin,coelenterazine, preferably a sulfated derivative thereof, or an analogthereof, and molecular oxygen [see, e.g., U.S. Pat. No. 5,023,181]. Theapoprotein and other constituents of the photoprotein andbioluminescence generating reaction can be mixed under appropriateconditions to regenerate the photoprotein and concomitantly have thephotoprotein produce light. Reconstitution requires the presence of areducing agent, such as mercaptoethanol, except for modified forms,discussed below, that are designed so that a reducing agent is notrequired [see, e.q., U.S. Pat. No. 5,093,240].

For use herein, it is preferred aequorin is produced using DNA, such asthat set forth in SEQ ID Nos. 5-13 and known to those of skill in theart or modified forms thereof. The DNA encoding aequorin is expressed ina host cell, such as E. coli, isolated and reconstituted to produce thephotoprotein [see, e.g., U.S. Pat. Nos. 5,418,155, 5,292,658, 5,360,728,5,422,266, 5,162,227].

Of interest herein, are forms of the apoprotein that have been modifiedso that the bioluminescent activity is greater than unmodifiedapoaequorin [see, e.g., U.S. Pat. No. 5,360,728, SEQ ID Nos. 10-12].Modified forms that exhibit greater bioluminescent activity thanunmodified apoaequorin include proteins having sequences set forth inSEQ ID Nos. 10-12, in which aspartate 124 is changed to serine,glutamate 135 is changed to serine, and glycine 129 is changed toalanine, respectively. Other modified forms with increasedbioluminescence are also available.

For use in certain embodiments herein, the apoprotein and othercomponents of the aequorin bioluminescence generating system arepackaged or provided as a mixture, which, when desired is subjected toconditions under which the photoprotein reconstitutes from theapoprotein, luciferin and oxygen [see, e.g., U.S. Pat. No. 5,023,181;and U.S. Pat. No. 5,093,240]. Particularly preferred are forms of theapoprotein that do not require a reducing agent, such as2-mercaptoethanol, for reconstitution. These forms, described, forexample in U.S. Pat. No. 5,093,240 [see, also Tsuji et al. (1986) Proc.Natl. Acad. Sci. U.S.A. 83:8107-8111], are modified by replacement ofone or more, preferably all three cysteine residues with, for exampleserine. Replacement may be effected by modification of the DNA encodingthe aequorin apoprotein, such as that set forth in SEQ ID No. 5, andreplacing the cysteine codons with serine.

The photoproteins and luciferases from related species, such as Obeliaare also contemplated for use herein. DNA encoding the Ca²⁺-activatedphotoprotein obelin from the hydroid polyp Obelia longissima is knownand available [see, e.g., Illarionov et al. (1995) Gene 153:273-274; andBondar et al. (1995) Biochim. Biophys. Acta 1231:29-32]. Thisphotoprotein can also be activated by Mn²⁺ [see, e.g., Vysotski et al.(1995) Arch. Bioch. Biophys. 316:92-93, Vysotski et al. (1993) J.Bio-lumin. Chemilumin. 8:301-305].

In general for use herein, the components of the bioluminescence arepackaged or provided so that there is insufficient metal ions to triggerthe reaction. When used, the trace amounts of triggering metal ion,particularly Ca²⁺ is contacted with the other components. For a moresustained glow, aequorin can be continuously reconstituted or can beadded or can be provided in high excess.

(2) Luciferin

The aequorin luciferin is coelenterazine and analogs therein, whichinclude molecules having the structure [formula (I)]:

in which R₁ is CH₂C₆H₅ or CH₃; R₂ is C₆H₅, and R₃ is p-C₆H₄OH or CH₃ orother such analogs that have activity. Preferred coelenterazine has the;tructure in which R¹ is p-CH₂C₆H₄OH, R₂ is C₆H₅, and R₃ is p-C₆H₄OH,which can be prepared by known methods [see, e.g., Inouye et al. (1975)Jap. Chem. Soc., Chemistry Lttrs. pp 141-144; and Halt et al. (1979)Biochemistry 18:2204-2210] The preferred coelenterazine has thestructure (formula (II)):

and sulfated derivatives thereof.

The reaction of coelenterazine wh en bound to the aequorin photoproteinwith bound oxygen and in the presence of Ca²⁺ can represented asfollows:

The photoprotein aequorin [which contains apoaequorin bound to acoelenterate luciferin molecule] and Renilla luciferase, discussedbelow, can use the same coelenterate luciferin. The aequorinphotoprotein catalyses the oxidation of coelenterate luciferin[coelenterazine]to oxyluciferin [coelenteramide] with the concomitantproduction of blue light [lambda_(max)=469 nm].

Importantly, the sulfate derivative of the coelenterate luciferin[lauryr-luciferin] is particularly stable in water, and thus may be usedin a coelenterate-like bioluminescence generating system. In thissystem, adenosine diphosphate (ADP) and a sulpha-kinase are used toconvert the coelenterazine to the sulphated form. Sulfatase is then usedto reconvert the lauryl-luciferin to the native coelenterazine. Thus,the more stable lauryl-luciferin is used in the item to be illuminatedand the luciferase combined with the sulfatase are added to theluciferin mixture when illumination is desired.

Thus, the bioluminescence generating system of Aequorea is particularlysuitable for use in the methods and apparatus herein. The particularamounts and the manner in which the components are provided depends uponthe selected assay, luciferase and anti ligand. This system can beprovided in lyophilized form, that will glow upon addition of Ca²⁺. Itcan be encapsulated, linked to matrices, such as porous glass, or in asa compositions, such as a solution or suspension, preferably in thepresence of sufficient chelating agent to prevent triggering thereaction. The concentration of the aequorin photoprotein will vary andcan be determined empirically. Typically concentrations of at least 0.1mg/l, more preferably at least 1 mg/l and higher, will be selected. Incertain embodiments, 1-10 mg luciferin/100 mg of luciferase will be usedin selected volumes and at the desired concentrations will be used.

b. The Renilla System

Representative of coelenterate systems is the Renilla system. Renilla,also known as sea pansies, are members of the class of coelenteratesAnthozoa, which includes other bioluminescent genera, such asCavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus.Bioluminescent members of the Anthozoa genera contain luciferases andluciferins that are similar in structure [see, e.g., Cormier et al.(1973) J. Cell. Physiol. 81:291-298; see, also Ward et al. (1975) Proc.Natl. Acad. Sci. U.S.A. 72:2530-2534]. The luciferases and luciferinsfrom each of these anthozoans crossreact and produce a characteristicblue luminescence.

Renilla luciferase and the other coelenterate and ctenophoreluciferases, such as the aequorin photoprotein, use imidazopyrazinesubstrates, particularlythe substrates generically called coelenterazine[see, formulae (I) and (II), above]. Other genera that have luciferasesthat use a coelenterazine include: squid, such as Chiroteuthis,Eucleoteuthis, Onychoteuthis, Watasenia; cuttlefish, Sepiolina; shrimp,such as Oplophorus, Sergestes, and Gnathophausia; deep-sea fish, such asArgyropelecus, Yarella, Diaphus, and Neoscopelus.

Renilla luciferase does not, however, have bound oxygen, and thusrequires dissolved oxygen in order to produce light in the presence of asuitable luciferin substrate. Since Renilla luciferase acts as a trueenzyme [i.e., it does not have to be reconstituted for further use] theresulting luminescence can be long-lasting in the presence of saturatinglevels of luciferin. Also, Renilla luciferase is relatively stable toheat.

Renilla luciferase, DNA encoding Renilla luciferase, and use of the DNAto produce recombinant luciferase, as well as DNA encoding luciferasefrom other coelenterates, are well known and available [see, e.g., SEQID No. 1, U.S. Pat. Nos. 5,418,155 and 5,292,658; see, also, Prasher etal. (1985) Biochem. Biophys. Res. Commun. 126:1259-1268; Cormier (1981)“Renilla and Aequorea bioluminescence” in Bioluminescence andChemiluminescence, pp. 225-233; Charbonneau et al. (1979) J. Biol. Chem.254:769-780; Ward et al. (1979) J. Biol. Chem. 254:781-788; Lorenz etal. (1981) Proc. Natl. Acad. Sci. U.S.A. 88: 4438-4442; Hori et al.(1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Hori et al. (1975)Biochemistry 14:2371-2376; Hori et al. (1977) Proc. Natl. Acad. Sci.U.S.A. 74:4285-4287; Inouye et al. (1975) Jap. Soc. Chem. Lett. 141-144;and Matthews et al. (1979) Biochemistry 16:85-91]. The DNA encodingRenilla luciferase and host cells containing such DNA provide aconvenient means for producing large quantities of the enzyme [see, e.g,U.S. Pat. Nos. 5,418,155 and 5,292,658, which describe recombinantproduction of Renilla luciferase and the use of the DNA to isolate DNAencoding other luciferases, particularly those from related organisms].A modified version of a method [U.S. Pat. Nos. 5,418,155 and 5,292,658]for the recombinant production of Renilla luciferase that results in ahigher level of expression of the recombinant enzyme is presented in theEXAMPLES herein.

When used herein, the Renilla luciferase can be packaged in lyophilizedform, encapsulated in a vehicle, either by itself or in combination withthe luciferin substrate. Prior to use the mixture is contacted with anaqueous composition, preferably a phosphate buffered saline or othersuitable buffer, such a Tris-based buffer [such as 0.1 mm Tris, 0.1 mmEDTA] pH 7-8, preferably about pH 8; dissolved O₂ will activate thereaction. Addition of glycerol [about 1%] increases light intensity.Final concentrations of luciferase in the glowing mixture will be on theorder of 0.01 to 1 mg/l or more. Concentrations of luciferin will be atleast about 10⁻⁸ M, but 1 to 100 or more orders of magnitude higher toproduce a long lasting bioluminescence.

In certain embodiments herein, about 1 to 10 mg, or preferably 2-5 mg,more preferably about 3 mg of coelenterazine will be used with about 100mg of Renilla luciferase. The precise amounts, of course can bedetermined empirically, and, also will depend to some extent on theultimate concentration and application. In particular, about addition ofabout 0.25 ml of a crude extract from the bacteria that express Renillato 100 ml of a suitable assay buffer and about 0.005 μg was sufficientto produce a visible and lasting glow [see, U.S. Pat. Nos. 5,418,155 and5,292,658, which describe recombinant production of Renilla luciferasel.Lyophilized mixtures, and compositions containing the Renilla luciferaseare also provided. The luciferase or mixtures of the luciferase andluciferin may also be encapsulated into a suitable delivery vehicle,such as a liposome, glass particle, capillary tube, drug deliveryvehicle, gelatin, time release coating or other such vehicle. Kitscontaining these mixtures, compositions, or vehicles and also a chipdevice and reagents for attaching biological molecules to the surface ofthe chip, are also provided. The luciferase may also be linked to ananti ligand through chemical or recombinant means for use in the methodsherein.

Recombinant Production of Renilla Reniformis Luciferase

The phagemid pTZ18R (Pharmacia) is a multi-purpose DNA vector designedfor in vitro transcriptions and useful for expression of recombinantproteins in bacterial hosts. The vector contains the bla gene, whichallows for the selection of transformants by resistance to ampicillin,and a polylinker site adjacent to the lacZ′ gene. The heterologous geneof interest is inserted in the polylinker and transcribed from the lacpromoter by induction, for example, withisopropyl-β-D-thiogalactopyranoside (IPTG).

The DNA encoding the Renilla reniformis luciferase has been cloned(e.g., see U.S. Pat. Nos. 5,292,658 and 5,418,155). The plasmidpTZRLuc-1 encodes the Renilla luciferase on a 2.2 Kbp EcoRI to Sstl DNAfragment inserted in EcoRI and Sstl sites of pTZ18R (plasmidconstruction is described U.S. Pat. Nos. 5,292,658 and 5,418,155; seealso Lorenz et al. (1991) Isolation and Expression of a cDNA encodingRenilla reniformis Luciferase, Proc. Natl. Acad. Sci. U.S.A. 88,4438-4442). The initiation of transcription of the Renilla luciferasecDNA is under the control of the lacZ′ promoter. E. coli strainsharboring plasmid pTZRLuc-1 express Renilla luciferase that isfunctional in bioluminescence assays and retains the properties of thenative enzyme (see e.g., U.S. Pat. Nos. 5,292,658 and 5,418,155).

A derivative of pTZRLuc-1, pTZRLuc-3.6, produces approximately 7-foldhigher levels of recombinant Renilla luciferase than pTZRLuc-1 whentransformed into the same E. coli host. Competent E. coli strain XL-1was transformed using purified pTZRLuc-3.6 according to the instructionsprovided by the manufacturer (XL-1 Supercompetent cells and protocol;Stratagene, Inc., La Jolla, Calif.). Transfectants were selected byplating on Luria Broth (LB) plates supplemented with 100 μg/mlampicillin.

Single ampicillin resistant colonies were grown in LB mediumsupplemented with 100 μg/ml ampicillin at ambient temperature usingcontinuous shaking until cell growth reached mid-log phase (i.e., cellculture reaches an O.D._(600nm)=0.6-0.8 units). Transcription from thelac promoter was induced by addition of 1 mM IPTG and cell culture wasshaken at ambient temperature for an additional 8 hours.

Cells were harvested by centrifugation at 10,000×g and frozen at −20° C.The cell pellet was thawed and resuspended at a 1:5 ratio (w/w) in asolution of 10 mM EDTA, pH 8.0, containing 4 mg/ml lysozyme (SigmaChemical Corp.). The cells were placed in a 25° C. water bath for 30minutes and then transferred to ice for 1 hour. The cells were lysed bysonication at 0° C. using a 1 minute pulse from an Ultrasonics, Inc.cell disruptor.

The lysed cellular debris was removed by centrifugation at 30,000×g for3 hours and the supernatant was decanted and retained. The pellet wasresuspended at a 1:5 ratio in the above-described solutions, and thesubsequent incubations, lysis and centrifugation steps were repeated.The two supernatants were combined and stored at −70° C.

The resulting “clarified lysate” was employed as a source of recombinantluciferase. Alternatively, the lysate may be subjected to additionalpurification steps (e.g., ion exchange chromatography or immunoaffinitychromatography) to further enrich the lysate or provide a homogeneoussource of the purified enzyme (see e.g., U.S. Pat. Nos. 5,292,658 and5,418,155).

3. Crustacean, Particularly Cyrpidina Systems

The ostracods, such as Vargula serratta, hilgendorfii and noctiluca aresmall marine crustaceans, sometimes called sea fireflies. These seafireflies are found in the waters off the coast of Japan and emit lightby squirting luciferin and luciferase into the water, where thereaction, which produces a bright blue luminous cloud, occurs. Thereaction involves only luciferin, luciferase and molecular oxygen, and,thus, is very suitable for application herein.

The systems, such as the Vargula bioluminescence generating systems, areparticularly preferred herein because the components are stable at roomtemperature if dried and powdered and will continue to react even ifcontaminated. Further, the bioluminescent reaction requires only theluciferin/luciferase components in concentrations as low as 1:40 partsper billion to 1:100 parts per billion, water and molecular oxygen toproceed. An exhausted system can renewed by addition of luciferin.

a. Vargula Luciferase

Vargula luciferase is a 555-amino acid polypeptide that has beenproduced by isolation from Vargula and also using recombinant technologyby expressing the DNA in suitable bacterial and mammalian host cells[see, e.g., Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6567-6571; Inouye et al. (1992) Proc. Natl. Acad. Sci. U.S.A.89:9584-9587; Johnson et al. (1978) Methods in Enzymology LVII:331-349;Tsuji et al. (1978) Methods Enzymol. 57:364-72; Tsuji (19740Biochemistry 13:5204-5209; Japanese Patent Application No. JP 3-30678Osaka; and European Patent Application No. EP 0 387 355 A1].

(1) Purification From Cypridina

Methods for purification of Vargula [Cypridina] luciferase are wellknown. For example, crude extracts containing the active can be readilyprepared by grinding up or crushing the Vargula shrimp. In otherembodiments, a preparation of Cypridina hilgendorfi luciferase can beprepared by immersing stored frozen C. hilgendorfi in distilled watercontaining, 0.5-5.0 M salt, preferably 0.5-2.0 M sodium or potassiumchloride, ammonium sulfate, at 0-30° C., preferably 0-10° C., for 1-48hr, preferably 10-24 hr, for extraction followed by hydrophobicchromatography and then ion exchange or affinity chromatography [TORAYIND INC, Japanese patent application JP 4258288, published Sep. 14,1993; see, also, Tsuji et al. (1978) Methods Enzymol. 57:364-72 forother methods].

The luciferin can be isolated from ground dried Vargula by heating theextract, which destroys the luciferase but leaves the luciferin intact[see, e.g., U.S. Pat. No. 4,853,327].

(2) Preparation by Recombinant Methods

The luciferase is preferably produced by expression of cloned DNAencoding the luciferase [European Patent Application No. 0 387 355 A1;International PCT Application No. WO 90/01542; see, also SEQ ID No. 5,which sets forth the sequence from Japanese Patent Application No. JP3-30678 and Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6567-6571] DNA encoding the luciferase or variants thereof isintroduced into E. coli using appropriate vectors and isolated usingstandard methods.

b. Vargula Luciferin

The natural luciferin in a substituted imidazopyrazine nucleus, such acompound of formula (III):

Analogs thereof and other compounds that react with the luciferase in alight producing reaction also may be used.

Other bioluminescent organisms that have luciferases that can react withthe Vargula luciferin include, the genera Apogon, Parapriacanthus andPorichthys.

c. Reaction

The luciferin upon reaction with oxygen forms a dioxetanone intermediate[which includes a cyclic peroxide similar to the firefly cyclic peroxidemolecule intermediate]. In the final step of the bioluminescentreaction, the peroxide breaks down to form CO₂ and an excited carbonyl.The excited molecule then emits a blue to blue-green light.

The optimum pH for the reaction is about 7. For purposes herein, any pHat which the reaction occurs may be used. The concentrations of reagentsare those normally used for analytical reactions or higher [see, e.g,Thompson et al (1990) Gene 96:257-262]. Typically concentrations of theluciferase between 0.1 and 10 mg/l, preferably 0.5 to 2.5 mg/l will beused. Similar concentrations or higher concentrations of the luciferinmay be used.

4. Insect Bioluminescence Generating Systems Including Firefly, ClickBeetle, and Other Insect Systems

The biochemistry of firefly bioluminescence was the firstbioluminescence generating system to be characterized [see, e.g.,Wienhausen et al. (1985) Photochemistry and Photobiology 42:609-611;McElroy et al. (1966) in Molecular Architecture in Cell Physiology,Hayashi et al., eds. Prentice Hall, Inc., Englewood Cliffs, N.J., pp.63-80] and it is commercially available [e.g., from Promega Corporation,Madison, Wis., see, e.g., Leach et al. (1986) Methods in Enzymology133:51-70, esp. Table 1]. Luciferases from different species offireflies are antigenically similar. These species include members ofthe genera Photinus, Photurins and Luciola. Further, the bioluminescentreaction produces more light at 30° C. than at 20° C., the luciferase isstabilized by small quantities of bovine albumin serum, and the reactioncan be buffered by tricine.

a. Luciferase

DNA clones encoding luciferases from various insects and the use toproduce the encoded luciferase is well known. For example, DNA clonesthat encode luciferase from Photinus pyralis, Luciola cruciata [see,e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7870-7873;de We et al. (1986) Methods in Enzymology 133:3; U.S. Pat. No.4,968,613, see, also SEQ ID No. 3] are available. The DNA has also beenexpressed in Saccharomyces [see, e.g., Japanese Application No. JP63317079, published Dec. 26, 1988, KIKKOMAN CORP] and in tobacco.

In addition to the wild-type luciferase modified insect luciferases havebeen prepared. For example, heat stable luciferase mutants, DNA-encodingthe mutants, vectors and transformed cells for producing the luciferasesare available. A protein with 60% amino acid sequence homology withluciferases from Photinus pyralis, Luciola mingrelica, L. cruciata or L.lateralis and having luciferase activity is available [see, e.g.,International PCT Application No. WO 95/25798]. It is more stable above30° C. than naturally-occurring insect luciferases and may also beproduced at 37° C. or above, with higher yield.

Modified luciferases that generate light at different wavelengths[compared with native luciferase], and thus, may be selected for theircolor-producing characteristics. For example, synthetic mutant beetleluciferase(s) and DNA encoding such luciferases that producebioluminescence at a wavelength different from wild-type luciferase areknown [Promega Corp, International PCT Application No. WO 95/18853,which is based on U.S. application Ser. No. 08/177,081 Jan. 3, 1994. Themutant beetle luciferase has an amino acid sequence differing from thatof the corresponding wild-type Luciola cruciata [see, e.g., U.S. Pat.Nos. 5,182,202, 5,219,737, 5,352,598, see, also SEQ ID No.3] by asubstitution(s) at one or two positions. The mutant luciferase producesa bioluminescence with a wavelength of peak intensity that differs by atleast 1 nm from that produced by wild-type luciferases.

Other mutant luciferase have also been produced. Mutant luciferases withthe amino acid sequence of wild-type luciferase, but with at least onemutation in which valine is replaced by isoleucine at the amino acidnumber 233, valine by isoleucine at 239, serine by asparagine at 286,glycine by serine at 326, histidine by tyrosine at 433 or proline byserine at 452 are known [see, e.g., U.S. Pat. Nos. 5,219,737, and5,330,906]. The luciferases are produced by expressing DNA-encoding eachmutant luciferase in E. coli and isolating the protein. Theseluciferases produce light with colors that differ from wild-type. Themutant luciferases catalyze luciferin to produce red [λ609 nm and 612nm], orange[λ595 and 607 nm] or green [λ558 nm] light. The otherphysical and chemical properties of mutant luciferase are substantiallyidentical to native wild type-luciferase. The mutant luciferase has theamino acid sequence of Luciola cruciata luciferase with an alterationselected from Ser 286 replaced by Asn, Gly 326 replaced by Ser, His 433replaced by Tyr or Pro 452 replaced by Ser. Thermostable luciferases arealso available [see, e.g., U.S. Pat. No. 5,229,285; see, alsoInternational PCT Application No.@) 95/25798, which provides Photinusluciferase in which the glutamate at position 354 is replaced Jysine andLuciola luciferase in which the glutamate at 356 is replaced withlysinel.

These mutant luciferases as well as the wild type luciferases are amongthose preferred herein, particularly in instances when a variety ofcolors are desired or when stability at higher temperatures is desired.It is also noteworthy that firefly luciferases have alkaline pH optima[7.5-9.5], and, thus, are suitable for use in diagnostic assaysperformed at alkaline pH.

b. Luciferin

The firefly luciferin is a benzothiazole:

Analogs of this luciferin and synthetic firefly luciferins are alsoknown to those of skill in art [see, e.g., U.S. Pat. No. 5,374,534 and5,098,828]. These include compounds of formula (IV) [see, U.S. Pat. No.5,098,828]:

in which:

R¹ is hydroxy, amino, linear or branched C₁-C₂₀ alkoxy, C₂-C₂₀alkyenyloxy, an L-amino acid radical bond via the α-amino group, anoligopeptide radical with up to ten L-amino acid units linked via theα-amino group of the terminal unit;

R² is hydrogen, H₂PO₃, HSO₃, unsubstituted or phenyl substituted linearor branched C₁-C₂₀ alkyl or C₂-C₂₀alkenyl, aryl containing 6 to 18carbon atoms, or R³—C(O)—; and

R³ is an unsubstituted or phenyl substituted linear or branched C₁-C₂₀alkyl or C₂-C₂₀alkenyl, aryl containing 6 to 18 carbon atoms, anucleotide radical with 1 to 3 phosphate groups, or a glycosidicallyattached mono- or disaccharide, except when formula (IV) is aD-luciferin or D-luciferin methyl ester.

c. Reaction

The reaction catalyzed by firefly luciferases and related insectluciferases requires ATP, Mg²⁺ as well as molecular oxygen. Luciferinmust be added exogenously. Firefly luciferase catalyzes the fireflyluciferin activation and the subsequent steps leading to the excitedproduct. The luciferin reacts with ATP to form a luciferyl adenylateintermediate. This intermediate then reacts with oxygen to form a cyclicluciferyl peroxy species, similar to that of the coelenterateintermediate cyclic peroxide, which breaks down to yield CO₂ and anexcited state of the carbonyl product. The excited molecule then emits ayellow light; the color, however, is a function of pH. As the pH islowered the color of the bioluminescence changes from yellow-green tored.

Different species of fireflies emit different colors of bioluminescenceso that the color of the reaction will be dependent upon the speciesfrom which the luciferase is obtained. Additionally, the reaction isoptimized at pH 7.8.

Addition of ATP and luciferin to a reaction that is exhausted producesadditional light emission. Thus, the system, once established, isrelatively easily maintained. Therefore, it is highly suitable for useherein in embodiments in which a sustained glow is desired or reuse ofthe item is contemplated. Thus, the components of a firefly system canbe packaged with the chip.

5. Bacterial Systems

Luminous bacteria typically emit a continuous light, usually blue-green.When strongly expressed, a single bacterium may emit 10⁴ to 10⁵ photonsper second. Bacterial bioluminescence systems include, among others,those systems found in the bioluminescent species of the generaPhotobacterium, Vibrio and Xenorhabdus. These systems are well known andwell characterized [see, e.g., Baldwin et al. (1984) Biochemistry23:3663-3667; Nicoli et al. (1974) J. Biol. Chem. 249:2393-2396; Welcheset al. (1981) Biochemistry 20:512-517; Engebrecht et al. (1986) Methodsin Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70; U.S.Pat. No. 4,581,335].

a. Luciferases

Bacterial luciferase, as exemplified by luciferase derived from Vibrioharveyi [EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase1-hydroxylating, luminescing], is a mixed function oxidase, formed bythe association of two different protein subunits α and β. The α-subunithas an apparent molecular weight of approximately 42,000 kD and theβ-subunit has an apparent molecular weight of approximately 37,000 kD[see, e.q., Cohn et al. (1989) Proc. Natl. Acad. Sci. U.S.A.90:102-123]. These subunits associate to form a 2-chain complexluciferase enzyme, which catalyzes the light emitting reaction ofbioluminescent bacteria, such as Vibrio harveyi [U.S. Pat. No.4,581,335; Belas et al. (1982) Science 218:791-793], Vibrio fischeri[Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et al. (1984)Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158] and other marine bacteria.

Bacterial luciferase genes have been cloned [see, e.g., U.S. Pat. No.5,221,623; U.S. Pat. No. 4,581,335; European Patent Application No. EP386 691 A]. Plasmids for expression of bacterial luciferase, such asVibrio harveyi, include pFIT001 (NRRL B-18080), pPALE001 (NRRL B-18082)and pMR19 (NRRL B-18081)] are known. For example the sequence of theentire lux regulon from Vibiro fisheri has been determined [Baldwin etal. (1984), Biochemistry 23:3663-3667; Baldwin et al. (1981) Biochem.20: 512-517; Baldwin et al. (1984) Biochem. 233663-3667; see, also,e.g., U.S. Pat. Nos. 5,196,318, 5,221,623, and 4,581,335]. This regulonincludes luxl gene, which encodes a protein required for autoinducersynthesis [see, e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci.U.S.A. 81:4154-4158], the luxC, luxD, and luxE genes, which encodeenzymes that provide the luciferase with an aldehyde substrate, and theluxA and luxB genes, which encode the alpha and beta subunits of theluciferase.

Lux genes from other bacteria have also been cloned and are available[see, e.g., Cohn et al. (1985) J. Biol. Chem. 260:6139-6146; U.S. Pat.No. 5,196,524, which provides a fusion of the luxA and luxB genes fromVibrio harveyi]. Thus, luciferase alpha and beta subunit-encoding DNA isprovided and can be used to produce the luciferase. DNA encoding the α[1065 bp] and β [984 bp] subunits, DNA encoding a luciferase gene of2124 bp, encoding the alpha and beta subunits, a recombinant vectorcontaining DNA encoding both subunits and a transformed E. coli andother bacterial hosts for expression and production of the encodedluciferase are available. In addition, bacterial luciferases arecommercially available.

b. Luciferins

Bacterial luciferins include:

in which the tetradecanal with reduced flavin mononucleotide areconsidered luciferin since both are oxidized during the light emittingreaction.

c. Reactions

The bacterial systems require, in addition to reduced flavin, fivepolypeptides to complete the bioluminescent reaction: two subunits, αand β, of bacterial luciferin and three units of a fatty acid reductasesystem complex, which supplies the tetradecanal aldehyde. Examples ofbacterial bioluminescence generating systems useful in the apparatus andmethods provided herein include those derived from Vibrio fisheri andVibrio harveyi. One advantage to this system is its ability to operateat cold temperatures. It will thus be particularly amenable to methodsof using the chip for the detection and monitoring of antibioticsensitivity of psychrophilic organisms. All components of a bacterialsystem can be frozen into ice or placed in solutions stored below 0° C.After incubation at temperatures near 0° C., the chip can be transferredto warmer temperatures and the reaction will proceed thereby providing asustained glow.

Bacterial luciferase catalyzes the flavin-mediated hydroxylation of along-chain aldehyde to yield carboxylic acid and an excited flavin; theflavin decays to ground state with the concomitant emission of bluegreen light [λ_(max)=490 nm; see, e.g., Legocki et al. (1986) Proc.Natl. Acad. Sci. USA 81:9080; see U.S. Pat. No. 5,196,524]:

The reaction can be initiated by contacting reduced flavinmononucleotide [FMNH₂] with a mixture of the bacterial luciferase,oxygen, and a long-chain aldehyde, usually n-decyl aldehyde.

DNA encoding luciferase from the fluorescent bacterium Alteromonashanedai is known [CHISSO CORP; see, also, Japanese application JP7222590, published Aug. 22, 1995]. The reduced flavin mononucleotide[FMNH₂; luciferin] reacts with oxygen in the presence of bacterialluciferase to produce an intermediate peroxy flavin. This intermediatereacts with a long-chain aldehyde [tetradecanal] to form the acid andthe luciferase-bound hydroxy flavin in its excited state. The excitedluciferase-bound hydroxy flavin then emits light and dissociates fromthe luciferase as the oxidized flavin mononucleotide [FMN] and water. Invivo FMN is reduced again and recycled, and the aldehyde is regeneratedfrom the acid.

Flavin reductases have been cloned [see, e.g., U.S. Pat. No. 5,484,723;see, SEQ ID No. 14 for a representative sequence from this patent].These as well as NAD(P)H can be included in the reaction to regenerateFMNH₂ for reaction with the bacterial luciferase and long chainaldehyde. The flavin reductase catalyzes the reaction of FMN, which isthe luciferase reaction, into FMNH₂; thus, if luciferase and thereductase are included in the reaction system, it is possible tomaintain the bioluminescent reaction. Namely, since the bacterialluciferase turns over many times, bioluminescence continues as long as along chain aldehyde is present in the reaction system.

The color of light produced by bioluminescent bacteria also results fromthe participation of a blue-florescent protein [BFP] in thebioluminescence reaction. This protein, which is well known [see, e.g.,Lee et al. (1978) Methods in Enzvmologc LVII:226-234], may also be addedto bacterial bioluminescence reactions in order to cause a shift in thecolor.

6. Other Systems

a. Dinoflagellate Bioluminescence Generating Systems

In dinoflagellates, bioluminescence occurs in organelles termedscintillons. These organelles are outpocketings of the cytoplasm intothe cell vacuole. The scintillons contain only dinoflagellate luciferaseand luciferin [with its binding protein], other cytoplasmic componentsbeing somehow excluded. The dinoflagellate luciferin is a tetrapyrrolerelated to chlorophyll:

or an analog thereof.

The luciferase is a 135 kD single chain protein that is active at pH6.5, but inactive at pH 8 [see, e.g., Hastings (1981) Bioluminescenceand Chemiluminescence, DeLuca et al., eds. Academic Press, N.Y.,pp.343-360]. Luminescent activity can be obtained in extracts made at pH8 by shifting the pH from 8 to 6. This occurs in soluble and particulatefractions. Within the intact scintillon, the luminescent flash occursfor ˜100 msec, which is the duration of the flash in vivo. In solution,the kinetics are dependent on dilution, as in any enzymatic reaction. AtpH 8, the luciferin is bound to a protein [luciferin binding protein]that prevents reaction of the luciferin with the luciferase. At pH 6,however, the luciferin is released and free to react with the enzyme.

b. Systems From Molluscs, Such as Latia and Pholas

Molluscs Latia neritoides and species of Pholas are bioluminescentanimals. The luciferin has the structure:

and has been synthesized [see, e.g., Shimomura et al. (1968)Biochemistry 7:1734-1738; Shimomura et al. (1972) Proc. Natl. Acad. Sci.U.S.A. 69:2086-2089]. In addition to a luciferase and luciferin thereaction has a third component, a “purple protein”. The reaction, whichcan be initiated by an exogenous reducing agent is represented by thefollowing scheme:

XH₂ is a reducing agent.

Thus for practice herein, the reaction will require the purple proteinas well as a reducing agent.

c. Earthworms and Other Annelids

Earthworm species, such as Diplocardea longa, Chaetopterus andHarmothoe, exhibit bioluminescence. The luciferin has the structure:

The reaction requires hydrogen peroxide in addition to luciferin andluciferase. The luciferase is a photoprotein.

d. Glow Worms

The luciferase/luciferin system from the glow worms that are found inNew Zealand caves, Australia and those found in Great Britain are alsointended for use herein.

e. Marine Polycheate Worm Systems

Marine polycheate worm bioluminescence generating systems, such asPhyxotrix and Chaetopterus, are also contemplated for use herein.

f. South American Railway Beetle

The bioluminescence generating system from the South American railwaybeetle is also intended for use herein.

g. Fish

Of interest herein, are luciferases and bioluminescence generatingsystems that generate red light. These include luciferases found inspecies of Aristostomias, such as A. scintillans [see, e.g., O'Day etal. (1974) Vision Res. 14:545-550], Pachystomias, Malacosteus, such asM. niger.

7. Fluorescent Proteins

a. Green and Blue Fluorescent Proteins

As described herein, blue light is produced using the Renilla luciferaseor the Aequorea photoprotein in the presence of Ca²⁺ and thecoelenterazine luciferin or analog thereof. This light can be convertedinto a green light if a green fluorescent protein (GFP) is added to thereaction. Green fluorescent proteins, which have been purified [see,e.g., Prasher et al. (1992) Gene 111:229-233] and also cloned [see,e.g., International PCT Application No. WO 95/07463, which is based onU.S. application Ser. No. 08/119,678 and U.S. application Ser. No.08/192,274, which are herein incorporated by reference], are used bycnidarians as energy-transfer acceptors. GFPs fluoresce in vivo uponreceiving energy from a luciferase-oxyluciferein excited-state complexor a Ca²⁺ -activated photoprotein. The chromophore is modified aminoacid residues within the polypeptide. The best characterized GFPs arethose of Aequorea and Renilla [see, e.g., Prasher et al. (1992) Gene111:229-233; Hart, et al. (1979)Biochemistry 18:2204-2210]. For example,a green fluorescent protein [GFP] from Aequorea Victoria contains 238amino acids, absorbs blue light and emits green light. Thus, inclusionof this protein in a composition containing the aequorin photoproteincharged with coelenterazine and oxygen, can, in the presence of calcium,result in the production of green light. Thus, it is contemplated thatGFPs may be included in the bioluminescence generating reactions thatemploy the aequorin or Renilla luciferases or other suitable luciferasein order to enhance or alter color of the resulting bioluminescence.

GFPs are activated by blue light to emit green light and thus may beused in the absence of luciferase and in conjunction with an externallight source with novelty items, as described herein. Similarly, bluefluorescent proteins (BFPs), such as from Vibrio fischeri, Vibrioharveyi or Photobacterium phosphoreum, may be used in conjunction withan external light source of appropriate wavelength to generate bluelight. (See for example, Karatani, et al., “A blue fluorescent proteinfrom a yellow-emitting luminous bacterium,” Photochem. Photobiol.55(2):293-299 (1992); Lee, et al., “Purification of a blue-fluorescentprotein from the bioluminescent bacterium Photobacterium phosphoreum”Methods Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); and Gast, etal. “Separation of a blue fluorescence protein from bacterialluciferase” Biochem. Biophys. Res. Commun. 80(1):14-21 (1978), each, asall references cited herein, incorporated in its entirety by referenceherein.) In particular, GFPs, and/or BFPs or other such fluorescentproteins may be used in the methods provided herein for the detection ofinfectious agents by binding an analyte to one or more anti ligand-GFPconjugate(s) at a plurality of locations and illuminating the chip withlight of an appropriate wavelength to cause the fluorescent proteins tofluoresce whereby the emitted fluorescence is detected by thephotodiodoes in the chip.

GFPs and/or BFPs or other such fluorescent proteins may be used inconjunction with any of the chips or devices described herein. Thesefluorescent proteins may also be used alone or in combination withbioluminescence generating systems to produce an array of colors. Theymay be used in combinations such that the color, for example, of theemitted light may be altered to maximize the amount of light availablefor detection by the photodiodes of the chip.

b. Phycobiliproteins

Phycobiliproteins are water soluble fluorescent proteins derived fromcyanobacteria and eukaryotic algae [see, e.q., Apt et al. (1995) J. Mol.Biol. 238:79-96; Glazer (1982) Ann. Rev. Microbiol. 36:173-198; andFairchild et al. (1994) J. of Biol. Chem. 269:8686-86941. These proteinshave been used as fluroescent labels in immmunoassay [see, Kronick(1986) J. of Immunolog. Meth. 92:1-13], the proteins have been isolatedand DNA encoding them is also available [see, e.g., Pilot et al. (1984)Proc. Natl. Acad. Sci. U.S.A. 81:6983-6987; Lui et al. (1993) PlantPhysiol 103:293-294; and Houmard et al. (1988) J. Bacteriol.170:5512-5521; the proteins are commercially available from, forexample, ProZyme, Inc., San Leandro, Calif.].

In these organisms, the phycobiliproteins are arranged in subcellularstructures termed phycobilisomes and function as accessory pigments thatparticipate in photosynthetic reactions by absorbing visible light andtransferring the derived energy to chlorophyll via a direct fluorescenceenergy transfer mechanism.

Two classes of phycobiliproteins are known based on their color:phycoerythrins (red) and phycocyanins (blue), which have reportedabsorbtion maxima between 490 and 570 nm and between 610 and 665 nm,respectively. Phycoerythrins and phycocyanins are heterogenous complexescomposed of different ratios of alpha and beta monomers to which one ormore class of linear tetrapyrrole chromophores are covalently bound.Particular phycobiliproteins may also contain a third y-subunit whichoften associated with (αβ)₆ aggregate proteins.

All phycobiliproteins contain phycothrombilin or phycoerythobilinchromophores, and may also contain other bilins, such as phycourobilin,cryptoviolin or a 697 nm bilin. The y-subunit is covalently bound withphycourobilin, which results in the 495-500 nm absorbance peak of B- andR-phycoerythrins. Thus, the spectral characteristics ofphycobiliproetins may be influenced by the combination of the differentchromophores, the subunit composition of the apo-phycobiliproteinsand/or the local enviroment that affects the tertiary and quaternarystructure of the phycobiliproteins.

As described above for GFPs & BFPs, phycobiliproteins are also activatedby visible light of the appropriate wavelength and thus may be used inthe absence of luciferase and in conjunction with an external lightsource to illuminate the phycobiliprotein bound to the chip at locationswhere analyte has been detected. In particular, phycobiliproteins may becovalently bound to one or more anti-ligand specific for the targetedanalyte and illuminated with light of an appropriate wavelength to causethe fluorescent proteins to fluoresce and the fluorescence is measuredby the photodiodes of the chip at that location of the array. The datasignals are sent to the commuter processor and analyzed. As noted above,these proteins may be used in combination with other fluoresent proteinsand/or bioluminescence generating systems to produce an array of colorsor to provide different colors over time that can be detected by thephotodiodes of the chip.

Attachment of phycobiliproteins to solid support matrices is known(e.g., see U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and4,867,908). Therefore, phycobiliproteins may be coupled to microcarrierscoupled to one or more components of the bioluminescent reaction,preferably a luciferase, to convert the wavelength of the lightgenerated from the bioluminescent reaction. Microcarriers coupled to oneor more phycobiliproteins may be used when linked to the anti-ligand orto any of the chips used in the methods herein.

C. Design, Fabrication, and Use of Chips

Chips for use as diagnostic devices are provided herein. The chips canbe nonself-addressable or self-addressable and are typically in the formof an array, such as a 96-member or higher density array or any of thosedescribed herein.

1. Nonself-addressable Chips

Referring to FIG. 1, a nonself-addressable microelectronic device 100for detecting and identifying analytes in a biological sample usingbioluminescence includes an address control circuit 102, a photodetectorarray 104, an analog multiplexer 106, a comparator 108, a referencecircuit 110, a feedback control circuit 112 and an output controlcircuit 114. Address control circuit 102 receives a clock input signal116 from an external oscillator, and output control circuit 114generates data output signals 118. Device 100 also includes electricalconnections 120 and 122 for receiving electrical power and ground,respectively, from an external power source (e.g., an AC-DC converter).Thus, device 100 requires only four electrical connections: clock inputsignal 116; data output signals 118; power 120; and ground 122.

Address control circuit 102 receives clock input signal 11 6 andgenerates address signals on busses 124-128 in response thereto whichsequentially address each pixel element within array 104. Each pixelelement has a row and a column address that are used to address thepixel. Address control circuit 102 sequentially addresses each row ofpixel elements within array 104 using row address signals asserted onbus 124. For each row, address control circuit 102 generates addresssignals on bus 126 that are used as select signals by analog multiplexer106, and also generates address signals on bus 128 that are used byfeedback control circuit 112 to generate feedback signals for the pixelelements as described below. Address control circuit 102 generatesbinary address signals decoded into individual row and column addressenable signals by one or more address decode circuits located in addresscontrol circuit 102, array 104, multiplexer 106 and/or feedback controlcircuit 112. The addressing of an array in electronic circuits is wellknown to those of ordinary skill in the art.

Array 104 receives row address signals 124 from address control circuit102 and feedback signals 130 from feedback control circuit 112. Eachelement in array 104 includes a photodetector that receives photons oflight from a chemical reaction optically coupled to the photodetector.Based on these inputs, array 104 generates analog column output signals132 that are applied to analog multiplexer 106. Array 104 uses rowaddress signals 124 to address each row of pixel elements, uses feedbacksignals 130 when performing a delta-sigma analog-to-digital (A/D)conversion on each pixel element as described below, and generatescolumn output signals 132 that are also used in the delta-sigma A/Dconversion.

Analog multiplexer 106 uses address signals 126 to multiplex columnoutput signals 132 into multiplexed analog output signals 134.Comparator 108 compares multiplexed output signals 134 to a referencesignal 136 (e.g., a reference current) generated by reference circuit110 and, based upon the results of the comparison, generates quantizedoutput signals 138. Quantized output signals 138 and address signals 128are used by feedback control circuit 112 to generate feedback signals130 that are applied to array 104 as described below. Quantized outputsignals 138, that are indicative of the photons of light detected ateach element in array 104, are also used by output control circuit 114to generate data output signals 118.

In one embodiment, output control circuit 114 formats quantized outputsignals 138 into an RS-232 serial data stream indicative of the lightdetected at each pixel element in array 104. To allow an externalinstrument or computer to correlate the received RS-232 serial datastream with specific pixel elements in array 104, output control circuit114 transmits the serial data stream in frames separated by asynchronization signal (sync). Each frame contains an output data signalfor each pixel element in array 104, and the sync signal is an arbitraryvalue (e.g., a byte having a value of decimal 255) used as a controlsignal to identify the start of each data frame. The external computerwaits for the sync signal before correlating the received frame data tothe appropriate pixel elements in array 104. Alternatively, outputcontrol circuit 114 could include labels in the serial data stream thatidentify the pixel elements. A parallel data interface can also be used.

As will become apparent from the description below, array 104 includespixel elements located at an array of micro-locations on the surface ofthe semiconductor substrate used for device 100. Each element includes aphotodetector for receiving photons of light emitted by a chemicalreaction optically coupled at the respective micro-location and forconverting the received photons into an electric charge. Each elementalso includes a pixel unit cell circuit with a capacitance circuit forintegrating the electric charge. The integrated charge is quantizedusing delta-sigma A/D conversion techniques, and the digitized signal ismultiplexed into a serial data output stream interfaced to an externalcomputer. The computer executes a control program to integrate thedelta-sigma digital signal for a desired integration period ranging fromseconds to hours depending on the desired resolution. In one embodiment,the delta-sigma AID conversion is clocked for a 56 Kbaud interface toachieve 12-bit resolution in an integration period of about 10 seconds,and 16-bit resolution in a time period of about 3 minutes.

Referring to FIG. 2, device 100 includes a semiconductor substrate ordie 140 having array 104 defined on a surface thereof. Array 104includes an array of micro-locations 142, and an independentphotodetector 144 optically coupled to each micro-location. (Only theleft-most micro-location 142 and photodetector 144 in each row arelabeled in FIG. 2 for clarity.) Array 104 includes three sub-arrays 146,148 and 150 having three different sizes of micro-locations 142.Sub-array 146 includes a 4×16 array of 50 micron square pixels,sub-array 148 includes a 2×8 array of 100 micron square pixels, andsub-array 150 includes a 1×4 array of 200 micron square pixels.Photodetectors 144 are located on a portion of the surface of die 140 ateach micro-location 142. The portion taken up by photodetector 144includes about 90% of the surface area for larger pixel elements andabout 50% for smaller pixel elements. In one embodiment, photodetectors144 are silicon photodiodes that convert photons of light impinging ontheir surfaces into a photocurrent. The quantum efficiency of thisconversion is about 40% at a wavelength of 500-800 nm (i.e., aphotocurrent of 40 electrons is generated for each 100 photons ofreceived light). Photodiodes 144 can thus convert low photon levels intomeasurable signals. The surface of substrate 140 has a slight depression(e.g., 1 micron) at each micro-location 142 to help contain the fluidsample applied to device 100.

Array 104 is formed on a relatively small die 140 (e.g., 2.4×2.4 mm) toallow for low-cost production of device 100. Die 140 also has theelectronic circuitry of device 100 formed thereon (not shown in FIG. 2),and the outer perimeter of die 140 includes bonding pads 152 thatconnect to the electronic circuitry via traces formed on the die. Pads152 are bonded by wire bonds or other conductors to external leads orconductors of the microelectronic package for die 140 as shown in FIG.3. Pads 152 include pads for clock input signal 116, data output signal118, electrical power 120, ground 122, and various test signals asdesired. In one embodiment, microelectronic device 100 is an integratedcircuit device fabricated using a standard CMOS process well known tothose of skill in the art.

The larger pixels elements (e.g., 200 um) in array 104 have a highersensitivity to detect lower concentrations of analytes than the smallerpixel elements (e.g., 50 um) since a greater number of receptorantibodies can be bound to their larger surface areas, as explainedherein, such that more photons of light will be emitted when a chemicalreaction occurs at the respective micro-location. The smaller pixels canbe used to form a larger matrix on a given die size to allow a greaternumber of assays to be performed simultaneously. The optimum pixel sizefor detecting a particular analyte may be determined empirically. Theuse of different-sized pixel elements on device 100 has two advantages.First, larger pixel elements can be used to detect analytes requiringlarger sensitivities while smaller pixel elements can be used toincrease the number of pixel elements in the matrix for analytes havinglower sensitivities. Second, different sizes can be used to helpdetermine the optimum size for a particular analyte by empiricaltesting, with the optimum size being used for other embodiments of array104.

Alternative arrangements of array 104 will be apparent to a person ofordinary skill in the art. For example, array 104 can include sub-arraysof pixel elements having different sizes (as in FIG. 2), or an arrayhaving only a single pixel size (e.g., a 12×16 array of 50 micronpixels). The size of each pixel (e.g., 50, 100, 200 microns in FIG. 2)can be modified (e.g., a 400 micron pixel can be used). Also, the numberof pixels in the array or sub-array (e.g., 4×16, 2×8 or 1×16 in FIG. 2)can be changed to include an nxm array or sub-array having n rows and mcolumns, n and m being integers. Further, shapes other than squares canbe used for each pixel element (e.g., rectangles or circles). The sizeof die 140 can be modified to accommodate the different arrangements ofarray 104, although use of a larger die may increase the cost of device100. Also, die 140 may include more or fewer bonding pads 152, providedthere are separate pads for clock input signal 116, data output signals118, electrical power 120 and ground 122 (FIG. 1).

Referring to FIGS. 3 and 3A, die 140 is packaged within a ceramic dualin-line package (DIP) 154 with 40 pins or leads 156. Four pins 156 arededicated to clock input signal 116, data output signals 118, electricalpower 120, and ground 122. The other pins 156 are used for test signals.Other microelectronic packages may also be used, such as plasticpackages having more or fewer conductors including the four requiredconductors.

Package 154 has a top layer 158 having an upper surface 160, a middlelayer 162 having an upper surface 164, and a lower layer 166 having anupper surface 168. Layers 158 and 162 are made of a non-conductivedielectric (em. ceramic) and lower layer 166 forms a conductive groundplane 170 electrically coupled to the ground pin 156 of package 154.Upper surface 160 of top layer 158 has a first square aperture 172formed therein. A ground conductor trace 174 formed on upper surface 160surrounds aperture 172 and is electrically coupled to ground by a groundtrace 176 also formed on upper surface 160 that passes through a cut 180formed in the outer perimeter of package 154, and attaches to groundplane 170. Aperture 172 reveals a square portion of middle layer 162having a second square aperture 182 formed therein. The dimensions ofsecond aperture 182 are smaller than the dimensions of first aperture172 such that a portion of upper surface 164 of middle layer 162 isvisible from the top of package 154. The visible portion of uppersurface 164 has conductive pads 184 formed thereon that electricallyconnect to pins 156 of package 154 via traces (only partially shown)passing between layers 158 and 162. Aperture 182 reveals a squareportion of ground plane 170 that has die 140 attached thereto by asuitable adhesive 186. Each bonding pad 152 of die 140 is electricallyconnected to one conductive pad 184 by a bond wire 188.

Bond wires 188 are coated with a material (e.g., epoxy) impervious tothe fluid sample to be analyzed. The other conductive components ofpackage 154, except for pins 156, may also be coated with the materialto prevent direct contact with the fluid sample. Pins 156 of package 154are not coated by the material such that pins 156 will make electricalcontact with an external computer or instrument when package 154 is readthereby.

When performing an assay, the fluid sample to be analyzed is appliedthrough apertures 172 and 182 to the surface of die 140 (andmicro-locations 142 formed thereon) housed within package 154. The fluidsample may be applied by pipetting the fluid sample into the test wellformed by apertures 172 and 182, or simply by dipping package 154 into acontainer (now shown) filled with the sample. The electrical componentsof device 100 are protected from the sample by the materials of package154 itself, or by the epoxy coating. After the fluid sample is applied,the remaining components needed to cause light-emitting reactionsoptically coupled to micro-locations 142 are also applied to the surfaceof die 140 through apertures 172 and 182. The resulting light-emittingreactions are then detected by photodetectors 144 as described below inrelation to FIG. 4.

Referring to FIG. 4, the photodetector 144 of each pixel element inarray 104 includes a pixel unit cell circuit 200 associated therewith.Each photodetector 144 is preferably a photodiode that generates sensedsignals (i.e., photocurrents) in response to photons of light 202impinging on its surface. Each pixel unit cell circuit 200 integratesthis sensed signal and quantizes the integrated signal using delta-sigmaA/D conversion techniques. Circuit 200 includes five MOSFET transistorsT₁-T₅ designated by numerals 204-212, each having a gate terminal G, asource terminal S (with an arrow pointing in toward the oxide layer), adrain terminal D, and a base terminal (unlabeled). Transistor T1 has itsgate G connected to a row enable input signal R_(en) designated 214, itssource S connected to power supply voltage V_(DD) and its drain Dconnected to source S of T₂. Transistor T₂ has its gate G connected to afeedback enable signal F_(en) designated 216, its source S connected todrain D of T₁, and its drain D connected to source S of T₃ at Node 3.Transistor T₃ has its gate G connected to a next row enabled signalR_(en1) designated 218, its source S connected to Node 3, and its drainD connected to gate G of T₄ at Node 1. The cathode of photodiode 144 isalso connected to Node 1, and its anode is connected to ground.Transistor T₄ has its gate G connected to Node 1, its source S connectedto V_(DD), and its drain D connected to source S of T₅, at Node 2.Transistor T₅ has its gate G connected to R_(en) (214), its source Sconnected to Node 2, and its drain D connected to output terminal 220.The base of each transistor T₁-T₅ is connected to V_(DD). Transistor T₂uses a second layer of polysilicon for its gate G to allow for aslightly smaller spacing between transistors, while transistors T₁ andT₃-T₅ use only a first layer of polysilicon.

The current flowing through photodiode 144, designated I_(D), includestwo components. The first component is a leakage current flowing throughphotodiode 144, that has a constant value. The second component is thecurrent flow caused by photons 202 impinging on photodiode 144 due tothe light-emitting chemical reaction, if any, optically coupled to therespective micro-location 142, taking into account the photodiode'squantum efficiency. Current I_(D) discharges Node 1 toward ground at arate depending on the leakage current and the number of photonsimpinging on photodiode 144. Node 1 will be discharged relativelyquickly when a large amount of light is received, and relatively slowlywhen little or no light is present. Even when no light is present, Node1 is still discharged due to the leakage component Of I_(D). Thephotocurrent component of I_(D) can be separated from the leakagecurrent component by taking dark readings when the light-emittingreactions are not occurring, taking test readings when the reactions aretaking place, and correcting the test readings using the dark readings(e.g., by subtracting the dark readings from the test readings). Thedark readings may be taken either before, after, or both before andafter, the actual test takes place.

Referring back to FIG. 1 for a moment, the output currents flowing fromoutput terminal 220, designated I_(OUT), are the column output signals132 that are multiplexed by analog multiplexer 106 to form multiplexedoutput signals 134 input to comparator 108. Comparator 108 maintainsI_(OUT) at a constant voltage since the sensed signal is a current, andgenerates quantized output signals 138 based upon comparisons betweensignals 134 and reference current 136. Feedback control circuit 112generates feedback signals 130, that form enable signals F_(en) (216),based upon quantized output signals 138 and address signals 128. F_(en)for each pixel element is generated during the time period when the nextpixel element is being addressed. When address control circuit 102addresses the next row of pixel elements, causing R_(en) (214) to beasserted for that row, the next row enabled signal Ren₁. (218) is alsoasserted for the previous row of pixel elements using address decodecircuits as are well known in the art.

Returning to FIG. 4, pixel unit cell circuit 200 operates as follows.Photons 202 impinging on photodiode 144 generate current I_(D) thatdischarges Node 1 at a rate depending on the number of photons of lightreceived, the photodiode's quantum efficiency, and the constant leakagecurrent. When this pixel element is addressed by address control circuit102 (i.e., R_(en) activated), transistors T₁ and T₅ are enabled (i.e.,become conductive). With T₅ conducting, and output terminal 220 held ata constant voltage less than V_(DD) by comparator 108, transistor T₄produces a current proportional to the difference between V_(DD)-V_(T)and the voltage at Node 1. V_(T) is the transistor threshold voltage(e.g., about 1 V). This current flows through transistor T₅ as I_(OUT).After passing through multiplexer 106, I_(OUT) is compared to referencecurrent 136 by comparator 108, which includes a differential currentamplifier. Quantized output signal 138 is reset to 0 by comparator 108when I_(OUT) is less than reference current 136 and is set to 1 whenI_(OUT) is greater than reference current 136.

When I_(OUT) is less than reference current 136 (i.e., quantized outputsignal 138=0), feedback control circuit 112 disables feedback signal 130(i.e., F_(en)=0) to keep transistor T₂ in a non-conducting state. Thus,the voltage at Node 3 is not affected and I_(D) continues to dischargeNode 1. When I_(OUT) exceeds reference current 136 (i.e., quantizedoutput signal 138=1), feedback control circuit 112 enables feedbacksignal 130 (i.e., F_(en)=1) to turn on T₂ while T₁ is still enabled byR_(en). This sets the capacitance (i.e., the inherent source and drainto bulk capacitance of the MOSFET transistors) at Node 3 to V_(DD).There will be no appreciable voltage drop across T₁. or T₂ since thesetransistors are turned on into their linear regions and no current isflowing. When the next row is addressed (causing R_(en1) to be set to1), the charge on the capacitance circuit is transferred to Node 1 toraise the voltage at Node 1. Thus, the capacitance circuit is reset toan initial charge whenever I_(OUT) transitions above reference current136. This charge transfer is standard in switched capacitor circuits,and is well known to those of skill in the art. Since I_(D) dischargesthe capacitance for a period of time before the discharge of Node 1 issufficient to trip comparator 108, the capacitance effectivelyintegrates I_(D) flowing through photodiode 144.

After being reset to its initial value, the capacitance at Node 1 isagain discharged by photodiode 144 at a rate dependent on the magnitudeof I_(D) until I_(OUT) again transitions above reference current 136, atwhich time Node 1 is again recharged. Thus, Node 1 is kept at a voltagenear the voltage value required for T₄ to produce reference current 136.The number of times that comparator 108 senses reference current 136exceeded (i.e., “comparator positive transitions”) is proportional tothe total charge that has flowed through photodiode 144. As statedpreviously, I_(D) is the sum of the constant leakage current and thecurrent due to sensed photons 202. Thus, the number of comparatorpositive transitions over a period of time can be used to acc-emittingreaction, after the number is adjusted for the leakage current flowingthrough photodiode 144 by subtracting the dark readings. Quantizedoutput signals 138, as stated above, are formatted into an RS-232 serialdata stream transmitted to an external computer as data output signals118.

Referring to FIG. 5, the voltages at Nodes 1, 2 and 3 during operationof device 100 are shown. The voltages at Nodes 1, 2 and 3 are designatedby curves 222, 224 and 226, respectively. The x-axis represents time(msec), and the y-axis represents voltage (V). Voltages at nodes 1 and 3are essentially equal during most of the downward sloping portions ofcurves 222 and 226, differing as shown in FIG. 5. At the start of eachcycle (i.e., at each comparator positive transition occurring at eachlarge spike in voltage at Node 3), the voltage at Node 1 is recharged toits initial value when Node 3 is set to V_(DD). Then, Node 1 isdischarged by I_(D) at a rate depending on the amount of light detectedby photodiode 144. A steep decreasing slope on curve 222 occurs whenphotodiode 144 receives a relatively large amount of light, while agradually decreasing slope occurs when photodiode 144 receivesrelatively little or no light. Current I_(OUT) caused by the differencebetween V_(DD)-V_(T) and the voltage at Node 1 is compared to referencecurrent 136 whenever the pixel is addressed (approximately every 0.1msec). When I_(OUT) is less than reference current 136, circuit 200integrates the sensed signal from photodiode 144 by continuing todischarge Node 1, and the voltage at Node 1 decreases as shown by curve222. When I_(OUT) exceeds reference current 136, F_(en) causes thecapacitance at Node 3 to be reset to V_(DD). Then, when the next row isaddressed (i.e., causing R_(en1) to be set), the charge on thiscapacitance circuit is transferred to Node 1, thereby raising thevoltage at Node 1. The cycle repeats throughout the integration timeperiod. The external computer counts the number of comparator positivetransitions that occur during the integration time period using dataoutput signal 118. After integration is complete, the computer correctsfor the leakage current using the dark readings. The corrected number ofcounts is proportional to the concentration of the analyte in the fluidsample.

Referring to FIG. 6, a system 300 for detecting and identifying analytesin a fluid sample using light-emitting reactions includes an adaptorcircuit board 302, a computer 304, an input device 306, and an outputdevice 308. System 300 forms a test instrument. Board includes azero-insertion force (ZIF) socket (not shown) for receiving device 100,housed in package 154, after it has been dipped into the fluid sample tobe analyzed and then exposed to the remaining components of thelight-emitting chemical reactions. Board 302 also includes an oscillatorcircuit 310 for generating clock input signal 116, and an AC-DC powersupply 312 for receiving AC power from an external AC power supply 314and for generating DC electrical power signal 120 therefrom. An RS-232serial data cable 316 carries serial data output signals 118 from board302 to computer 304.

Computer 304 includes a processing circuit 318, a memory circuit 320,and a serial interface circuit 322. Processing circuit 318 includes acentral processing unit such as a microprocessor or microcontroller thatreceives input signals 324 from input device 306 and transmits outputsignals 326 to output device 308 via I/O interface circuits (not shown).Memory circuit 320 includes three memory areas 328-332 includingvolatile and non-volatile memory. Memory area 328 stores the controlprogram executed by processing circuit 318 and the fixed and variabledata (e.g., calibration and empirical testing data) needed duringexecution. Optional memory area 330 stores an analyte map used byprocessing circuit 318 to identify the particular analyte being testedfor at each micro-location 142 in array 104. When the map is present,processing circuit 318 may be programmed to identify analytes detectedin the fluid sample by correlating the received data output signals 118to the analytes identified in the map, and to generate output signals326 to identify the detected analytes on output device 308. Memory area332 stores a data acquisition array used by processing circuit 318 toaccumulate the comparator positive transitions for each pixel elementduring the integration time period. The number of comparator positivetransitions received during this period is indicative of the amount oflight received by the photodetector 144 at each pixel element.

Input device 306 includes, for example, a keyboard, a mouse, a touchscreen, or another input device for generating input signals 324 used tocontrol operation of system 300. Input signals 324 from device 306 allowthe user to, for example, start and stop operation of system 300, inputanalyte map data, input a desired integration time period, and input anyother data or commands needed by processing circuit 318 to detect andidentify analytes in the fluid sample being analyzed. Input signals 324may also be used to configure computer 304 to read a particular device100 having a predetermined arrangement of array 104. Output device 308may include an electronic display for displaying the presence and/orconcentration of analytes in the fluid sample being analyzed in responseto output signals 326. Output device 308 may also include a printer fordisplaying such data.

In one embodiment, the user enters a desired integration time periodinto computer 304 using input device 306 before starting a test. Forexample, the user may input a period of 10 seconds for 12-bitresolution, or 3 minutes for 16-bit resolution. The user then appliesthe fluid sample to be analyzed to device 100 (e.g., by dunking package154 into a container holding the sample), adds the remaining componentsof the light-emitting reaction, and inserts package 154 into the ZIFsocket on board 302. Device 100 will then start to transmit frames ofdata over cable 316 to computer 304. Each frame includes the quantizeddelta-sigma A/D conversion data for each pixel element in array 104.Processing circuit 318 waits for the sync byte to determine the start ofa data frame. Once a frame starts, processing circuit 318 correlates thedata received in each frame with micro-locations 142 in array 104 (basedupon the known arrangement of array 104), and integrates the output datasignals 118 correlated with each micro-location 142 by accumulating thecomparator positive transitions in the respective locations in dataacquisition array 332. The transitions are accumulated for the durationof the desired integration time period. After the integration period iscomplete, processing circuit 318 corrects the integrated data to correctfor the leakage current through photodetectors 144 based upon darkreadings previously taken (e.g., by inserting package 154 into board 302before starting the light-emitting reactions). At this point, thecorrected data in each location of array 332 is related to the presenceof the analytes in the fluid sample being tested for. Processing circuit318 then generates output signals 326 that, when applied to outputdevice 308, causes output device 308 to display the corrected data. Thiscorrected data is related to the presence and/or concentration of eachanalyte being tested for by relationships determined empirically usingknown concentrations of analytes.

In another embodiment, the optional analyte map has been pre-programmedin memory area 330 with the identities of the analytes being tested forat each micro-location 142 in array 104 before the test is started(possibly by using input device 306). Then, instead of simply outputtingthe corrected data for display on output device 308, processing circuit318 performs the additional step of correlating the locations in dataacquisition array 332 with the analyte map to identify the analytes, andgenerates output signals 326 to identify that analytes the correcteddata represents.

In yet another embodiment, the data stored in memory area 328 includesthreshold data indicative of the presence of each analyte in the fluidsample being analyzed. The threshold data for each analyte may have beendetermined by empirical testing using a fluid sample having a knownminimum concentration of the analyte, or may simply be stored as anoffset from the dark readings. Processing circuit 318 then compares thecorrected data to the threshold data (or the uncorrected data to thedark readings when offsets are used) to determine which analytes arepresent in the fluid sample being analyzed. Output signals 326 are thengenerated so that the analytes present in the fluid sample are displayedor printed. This embodiment may also include the use of the analyte mapto allow processing circuit 318 to identify the analytes whose presencein the sample fluid is detected.

In still another embodiment, the data within memory area 328 includesempirically-determined equations, curves or tables representingrelationships between the corrected data and the concentrations of theanalytes being tested for. Methods for determining such equations,curves or tables are well known in the art, and can include computercurve-fitting techniques. Processing circuit 318 uses the corrected dataas input data for the equations, curves or tables to determine theconcentration of each analyte in the sample. Output signals 326 aregenerated so that the concentration of analytes present in the sampleare displayed or printed. This embodiment may again include the analytemap to allow processing circuit 318 to identify the analytes whoseconcentration in the sample fluid was determined.

System 300 provides a kit useful for evaluating device 100. This systemrequires the user to directly handle package 154, which may result inmechanical damage to pins 156 or electrostatic discharge damage tocircuit 100. To avoid the need for direct handling by the user, device100 may be mounted on a disposable test circuit board 400 as shown inFIG. 7. Device 100 may again be packaged in ceramic DIP package 154, ormay alternatively be packaged in another style of microelectronicpackage 402 (e.g., a leadless chip carrier) mounted on board 400.Varieties of microelectronic packages are well known in the art. Package402 is adhered (e.g., soldered) to board 400 such that the user onlyneeds to handle board 400, and does not need to handle package 402directly.

Package 402 includes leads or pins 156 that are electrically coupled totraces 116-122 formed on board 400 for the clock input signal, dataoutput signal, power and ground, respectively. Package 402 may have onlythese four pins to reduce cost in high-volume applications. Traces116-122 are electrically coupled to a cable 404 that attaches to aconnector 406, which attaches to a mating connector on the testinstrument or computer. The conductors of package 402, traces 116-122and the surface of board 400 are protected from the fluid sample to beanalyzed with an epoxy coating 408. Coating 408 is not applied overapertures 172 and 182 or die 140 to allow the fluid sample and theremaining components of the light-emitting reactions to be applied todevice 100.

Alternatively, multi-well chips are composed of three layers [see, e.g.,FIGS. 8-11]. The bottom layer forms the lower section of each well andincorporates a semiconductor layer, a photodiode at the bottom of eachwell and an anode electrode, i.e., metal wire surrounding each well. Themiddle layer fits into grooves in the bottom layer and is composed of areflective metal layer, an insulating layer, preferably derivatizedplastic or silicon, such as MYLAR (oriented polyethylene terephthalateis commercially available from the E.I. du Pont de Nemours & Co., Inc.)to which the specific antibody or ligand for each well is attached[e.g., antibodies attached to MYLAR; see FIG. 10]. The top cap layerforms the remaining upper portion of each well and also contains thecathode electrode. Analytes or reactants may be transported within oramong wells by free field electrophoresis by supplying direct current,or by reversing the polarity of the current, through the upper cathodeand lower anode [e.g., see FIG. 11].

When used, the chip is contacted with a sample and washed thoroughly.Buffer or other suitable compositions is added to each well, until thelevel is above the cathode position. The chip is then contacted with acomposition containing a luciferin or, preferably a luciferase,conjugated or fused or otherwise linked to an antibody or antibodybinding portion thereof or a plurality of such fusions. The antibodiesor portions thereof are each specific for the antigens of interest. Theremaining components of the bioluminescence generating system are addedand the chip is attached to a power source through a wire harness [see,e.g., see FIG. 11, bottom]. Light produced is contained within each welland is detected by the photodiode located at the bottom of each well.The reflective surface will enhance the signal. The detected signal isrelayed to a computer processing unit essentially as described above andthe computer identifies the detected well and then displays the specificinfectious agent detected on an accompanying monitor or printout [see,e.g., FIG. 20].

2. Self Addressable Chips

The self-addressable chips [see, e.g., FIGS. 12-16] include a matrix, aninsulating a layer, a metal layer to which an attachment layer and apermeation layer are affixed. The chip also includes photodiodes thatwill detect emitted light.

a. Matrix Materials

Any matrix or chip may be employed as a substrate for fabricating thedevices provided herein. The substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, etc. Thesubstrate may have any convenient shape, such as a disc, square, sphere,and a circle. The substrate and its surface preferably form a rigidsupport on which to carry out the reactions described herein. Thesubstrate and its surface should also be chosen to provide appropriatelight-absorbing characteristics. For instance, the substrate may be apolymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs,GaP, SiO₂, SiN₄, modified silicon, or any one of a wide variety ofpolymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,or combinations thereof. Other substrate materials will be readilyapparent to those of skill in the art in light of the discisoure herein.Presently preferred are silica substrates used in the fabrication ofmicroelectric chip devices.

b. Fabrication Procedures

i. Microlithography

International patent application Publication Nos. WO 95/12808 and WO96/07917 describe general microlithographic or photolithographictechniques that can be used for the fabrication of the complex “chip”type device that has a large number of small micro-locations. While thefabrication of devices does not require complex photolithography, theselection of materials and the requirement that an electronic devicefunction actively in aqueous solutions does not require specialconsiderations.

The 64 micro-location device shown in FIG. 14 of WO 95/12808 that can befabricated using relatively simple mask design and standardmicrolithographic techniques. Generally, the base substrate materialwould be a 1 to 2 centimeter square silicon wafer or a chipapproximately 0.5 millimeter in thickness. The silicon chip is firstovercoated with a 1 to 2 μm thick silicon dioxide (SiO₂) insulationcoat, which is applied by plasma enhanced chemical vapor deposition(PECVD).

The chips are preferably designed to contain detector elements, e.g.,photodiodes, that are incorporated into the semicondutor layer andcoupled through optical paths, such as by waveguides or other means, tothe other optical paths of the chip. In preferred embodiments, thedetector element is comprised of a linear array of photodiodes with anapproximate resolution of 1-5 microns, preferably 1-2 microns. Using adetector located with the chip, identification of a target in a testsample may be achieved at the site of the attachment of the biologicalmolecule or anti-ligand.

In the next step, a 0.2 to 0.5 μm metal layer (e.g., aluminum) isdeposited by vacuum evaporation. In addition to aluminum, suitablemetals for circuitry include gold, silver, tin, copper, platinum,palladium, carbon, and various metal combinations. Special techniquesfor ensuring proper adhesion to the insulating substrate materials(SiO₂) are used with different metals.

The chip is next overcoated with a positive photoresist (Shipley,Microposit AZ 1350 J), masked (light field) with the circuitry pattern,exposed and developed. The photosolubilized resist is removed, and theexposed aluminum is etched away. The resist island is now removed,leaving the aluminum circuitry pattern on the chip. This includes anoutside perimeter of metal contact pads, the connective circuitry(wires), and the center array of micro-electrodes thatserve as theunderlying base for the addressable micro-locations.

Using PECVD, the chip is overcoated first with a 0.2 to 0.4 micron layerof SiO₂, and then with a 0.1 to 0.2 micron layer of silicon nitride(Si₃N₄). The chip is then covered with positive photoresist, masked forthe contact pads and micro-electrode locations, exposed, and developed.Photosolubilized resist is removed, and the SiO_(2 and Si) ₃N₄ layersare etched way to expose the aluminum contact pads and micro-electrodes.The surrounding island resist is then removed, the connective wiringbetween the contact pads and the micro-electrodes remains insulated bythe SiO₂ and Si₃N₄ layers.

The SiO₂ and Si₃N₄ layers provide important properties for thefunctioning of the device. First, the second SiO₂ layer has bettercontact and improved sealing with the aluminum circuitry. It is alsopossible to use resist materials to insulate and seal. This preventsundermining of the circuitry due to electrolysis effects when themicro-electrodes are operating. The final surface layer coating of Si₃N₄is used because it has much less reactivity with the subsequent reagentsused to modify the micro-electrode surfaces for the attachment ofspecific binding entities.

At this point the micro-electrode locations on the device are modifiedwith a specialized permeation and attachment layer, which is a crucialelement required for the active functioning of the device. The objectiveis to create on the micro-electrode an intermediate permeation layerwith selective diffusion properties and an attachment surface layer withoptimal binding properties. The attachment layer should preferably havefrom 10⁵ to 10⁷ functionalized locations per square micron (μm²) for theoptimal attachment of specific binding entities. The attachment ofspecific binding entities must not overcoat or insulate the surface topercent the underlying micro-electrode from functioning. A functionaldevice requires some fraction (˜5% to 25%) of the actual metalelectro-electrode surface to remain accessible to solvent (H₂O)molecules, and to allow the diffusion of counter-ions (e.g., Na⁺ andCl⁻) and electrolysis gases (e.g., O₂ and H₂) to occur.

The intermediate permeation layer must also allow diffusion to occur.Additionally, the permeation layer should have a pore limit propertythatinhibits or impedes the larger binding entities, reactants, andanalytes from physical contact with the micro-electrode surface. Thepermeation layer keeps the active micro-electrode surface physicallydistinct from the binding entity layer of the micro-location.

In terms of the primary device function, this design allows theelectrolysis reactions required for electrophoretic transport to occuron micro-electrode surface, but avoids adverse electrochemical effectsto the binding entities, reactants, and analytes.

One preferred procedure for the derivatization of the metalmicro-electrode surface uses aminopropyltriethoxy silane (APS). APSreacts readily with the oxide and/or hydroxyl groups on metal andsilicon surfaces. APS provides a combined permeation layer andattachment layer, with primary amine groups for the subsequent covalentcoupling of binding entities. In terms of surface binding sites, APSproduces a relatively high level of functionalization (i.e., a largenumber of primary amine groups) on slightly oxidized aluminum surfaces,an intermediate level of functionalization on SiO₂ surfaces, and verylimited functionalization of Si₃N₄ surfaces, and very limitedfunctionalization of Si₃N₄ surfaces.

The APS reaction is carried out by treating the whole device (e.g., achip) surface for 30 minutes with a 10% solution of APS in toluene at50° C. The chip is then washed in toluene, ethanol, and then dried forone hour at 50° C. The micro-electrode metal surface is functionalizedwith a large number of primary amine groups (10⁵ to 10⁶ per squaremicron). Binding entities can now be covalently bound to the derivatizedmicro-electrode surface.

ii. Micromachining International patent application Publication Nos. WO95/12808 and WO 96/07917 describe micro-machining techniques (e.g.,drilling, milling, etc.) and non-lithographic techniques to fabricatedevices. In general, the resulting devices have relatively largermicro-locations (>100 microns) than those produced by microlithography.These devices could be used for analytical applications, as well as forpreparative type applications, as well as for preparative typeapplications, such as biopolymer synthesis. Large addressable locationscould be fabricated in three dimensional formats (e.g., tubes orcylinders) in order to carry a large amount of binding entities. Suchdevices could be fabricated using a variety of materials including, butnot limited to, plastic, rubber, silicon, glass (e.g., microchannelled,microcapillary, etc.), or ceramics. In the case of micromachineddevices, connective circuitry and large lectrode structures can beprinted onto materials using standard circuit oard printing techniquesknown to those skilled in the art.

In the instant application, the chips are preferably designed to containdetector elements, e.q., photodiodes, that are incorporated into thesemicondutor layer and coupled through optical paths, such as bywaveguides or other means, to the other optical paths of the chip. Inpreferred embodiments, the detector element is comprised of a lineararray of photodiodes with an approximate resolution of 1-5 microns,preferably 1-2 microns. Using a detector located with the chip,identification of a target in a test sample may be achieved at the siteof the attachment of the biological molecule or anti-ligand.

Addressable micro-location devices can be fabricated relatively easilyusing micro-machining techniques. FIG. 15 of WO 95/12808 shows aschematic of a representative 96 micro-location device. Thismicro-location device is fabricated from a suitable material stock (2cm×4 cm×1 cm), by drilling 96 proportionately spaced holes (1 mm indiameter) through the material. An electrode circuit board is formed ona thin sheet of plastic material stock, which fit precisely over the topof the micro-location component. The underside of the circuit boardcontains the individual wires (printed circuit) to each micro-location.Short platinum electrode structures (˜3-34 mm) are designed to extenddown into the individual micro-location chambers. The printed circuitwiring is coated with a suitable water-proof insulating material. Theprinted circuit wiring converges to a socket, which allows connection toa multiplex switch controller and DC power supply. The device ispartially immersed and operates in a common buffer reservoir.

While the primary function of the micro-locations in devices fabricatedby micro-machining and microlithography techniques is the same, theirdesigns are different. In devices fabricated by microlithography, thepermeation and attachment layers are formed directly on the underlyingmetal micro-electrode. In devices fabricated by micro machiningtechniques, the permeation and attachment layers are physicallyseparated from their individual metal electrode structure by a buffersolution in the individual chamber of reservoir. In micro-machineddevices the permeation and attachment layers can be formed usingfunctionalized hydrophilic gels, membranes, or other suitable porousmaterials.

In general, the thickness of the combined permeation and attachmentlayers ranges from 10 μm to 10 mm. For example, a modified hydrophilicgel of 26% to 35% polyacrylamide (with 0.1% polylysine), can be used topartially fill (˜0.5 mm) each of the individual micro-location chambersin the device. This concentration of gel forms an ideal permeation layerwith a pore limit of from 2 nm to 3 nm. The polylysine incoroporatedinto the gel provides primary amine functional groups for the subsequentattachment of specific binding entities. This type of gel permeationlayer allows the electrodes to function actively in the DC mode. Whenthe electrode is activated, the gel permeation layer allows smallcounter-ions to pass through it, but the larger specific binding entitymolecules are concentrated on the outer surface. Here they becomecovalently bonded to the outer layer of primary amines, whicheffectively becomes the attachment layer.

An alternative technique for the formation of the permeation andattachment layers is to incorporate into the base of each micro-locationchamber a porous membrane material. The outer surface of the membrane isthen derivatized with chemical functional groups to form the attachmentlayer. Appropriate techniques and materials for carrying out thisapproach are known to those skilled in the art.

The above description for the design and fabrication of a device shouldnot be considered as a limit to other variations or forms of the basicdevice. Many variations of the device with larger or smaller numbers ofaddressable micro-locations are envisioned for different analytical andpreparative applications. Variations of the device with largeraddressable locations are envisioned for preparative biopolymersynthesis applications. Variations are also contemplated aselectronically addressable and controllable reagent dispensers for usewith other devices, including those produced by microlithographictechniques.

C. Self-addressing of Chips

The chips and devices described in International patent applicationPublication Nos. WO 95/12808 and WO 96/07917 are able to electronicallyself-address each micro-location with a specific binding entity. Thedevice itself directly affects or causes the transport and attachment ofspecific binding entities to specific micro-locations. The deviceself-assembles itself in the sense that no outside process, mechanism,or equipment is needed to physically direct, position, or place aspecific binding entity at a specific micro-location. Thisself-addressing process is rapid and specific, and can be carried out ineither a serial or parallel manner.

A device can be serially addressed with specific binding entities bymaintaining the selected micro-location in a DC mode and at the oppositecharge (potential) to that of a specific binding entity. All othermicro-locations are maintained at the same charge as the specificbinding entity. In cases where the binding entity is not in excess ofthe attachment sites on the micro-location, it is necessary to activateonly one other micro-electrode to affect the electrophoretic transportto the specific micro-location. The specific binding entity is rapidlytransported (in a few seconds, or preferably less than a second) throughthe solution, and concentrated directly at the specific micro-locationwhere it immediately becomes covalently bonded to the special surface.The ability to electronically concentrate reactants or analytes (70) ona specific micro-location (72) is shown in FIG. 7 of the patent. Allother micro-locations remain unaffected by that specific binding entity.Any unreacted binding entity is removed by reversing the polarity ofthat specific micro-location, and electro-phoresing it to a disposallocation. The cycle is repeated until all desired micro-locations areaddressed with their specific binding entities. FIG. 8 of the patentshows the serial process for addressing specific micro-locations (81,83, 85) with specific oligonucleotide binding entities (82, 84, 86).

The parallel process for addressing micro-locations simply involvessimultaneously activating a large number (particular group or line) ofmicro-electrodes so that the same specific binding entity istransported, concentrated, and reacted with more than one specificmicro-locations.

When used, the chip is contacted with a sample, such as a body fluid,particularly urine, sputum or blood. The chip is then contacted with acomposition containing a luciferin or, preferably a luciferase,conjugated or linked or fused to an antibody or antibody binding portionthereof or a plurality of such fusions. The antibodies or portionsthereof are each specific for the antigens of interest. Detection iseffected by reacting the chip with a bioluminescence generating systemthat generates light detected by the photodiodes.

3. Attachment of Biological Molecules to the Surface of Chips

A large variety of methods are known for attaching biological molecules,including proteins, nucleic acids and peptide nucleic acids, to solidsupports [see. e.g., Affinity Techniques. Enzyme Purification: Part B.Methods in Enzymol., Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad. Press,N.Y. (1974) and Immobilized Biochemicals and Affinity Chromatography,Advances in Experimental Medicine and Biology, vol. 42, ed. R. Dunlap,Plenum Press, N.Y. (1974); U.S. Pat. No. 5,451,683, see, also U.S. Pat.Nos. 5,624,711, 5,412,087, 5,679,773, 5,143,854], particularly siliconchips are known.

These methods typically involve derivatization of the solid support toform a uniform layer of reactive groups on the support surface andsubsequent attachment of the biological molecule to the derivatizedsurface via a covalent bond between the reactive group and a reactivemoiety present on the biological molecule. Presently preferred methodsare those applicable for the derivatization and attachment of biologicalmolecules to silica substrates, particularly methods for derivatizingthe silica surface of microelectronic chip devices.

a. Derivatization of Silica Substrates

Numerous methods for derivitizing silica surfaces or for coatingsurfaces with silica and then derivatizing the surface are known. Anumber of reagents may be used to derivatize the surface of a silicasubstrate. For example, U.S. Pat. No. 4,681,870 describes a method forintroducing free amino or carboxyl groups onto a silica matrix.Alternatively, a layer of free amino groups or carboxyl groups may beintroduced using amino- and carboxymethyl silane derivatives, such as3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane,4-amino-butyltriethoxysilane,(aminoethylaminomethyl)phenethyltrimethoxysilane,2-(carbomethoxy)ethyltrichlorosilane,(10-carbomethoxydecyl)dimethyl-chlorosilane and2-(carbomethoxy)ethylmethyldichlorosilane (e.g., see Hulls Catalog).

The silica surface may also be derivatized to introduce a layer ofhydroxyl groups using alkyl- and alkoxyalkyl halogenated silanederivatives. The alkoxy groups of trialkoxysilanes are hydrolyzed totheir corresponding silanol species, which may occur during the formalpreparation of aqueous solutions or the reaction of the silane with theabsorbed moisture on the silica substrate. The silanols usually condensewith themselves or with alkoxysilanes to form siloxanes. Thesilanol-containing species are highly reactive intermediates that reactwith the hydroxyl groups on the surface of the silica [e.g., see Mohsenet al. (1995) J. Oral Rehabil. 22:213-220]. Furthermore, a silica matrixmay also be activated by treatment with a cyanogen halide under alkalineconditions. The anti-ligand is covalently attached to the surface uponaddition to the activated surface.

The selection and use of an appropriate derivatizing reagent is withinthe skill of the skilled artisan. For example, the selection of theappropriate silane derivative may be accomplished by empiricalevaluation of silanes within the predicted categories. In preparingthese silica substrates, the entire surface of the substrate may bederivatized with the appropriate silane derivative or the surface can bederivatized at only plurality of locations to form a discrete array. Thereagents and solutions containing biological molecules may be added tothe surface of the silica manually or by using a tamping tool or anyother tool known to those of skill in the art for this purpose.

b. Attachment of Biological Molecules

The attachment of biological molecules to the surface of silicasubstrates may be effected using procedures and techniques known in theart and described herein. The attachment of the biological molecule mayalso be effected in the absence or presence of a linker moiety (e.g.,see Section D1 below). Any linker known to those of skill in the art maybe used herein.

Derivatized silica substrates containing a layer of free amino orcarboxyl groups or other suitable group may subsequently be covalentlylinked to a free carboxyl or amino group on a heterobifunctional linkeror a biological molecule, such as a protein, a protein nucleic acid orother anti-ligand, in the presence of a carbodiimide. The use ofcarbodiimides [e.g., N-ethyl-N′-(y-dimethylaminopropylcarbodiimide], ascoupling agents is well known to those of skill in the art [see, e.g.,Bodansky et al. in “The Practice of Peptide Synthesis,” Springer-Verlag,Berlin (1984)].

Another method for attaching biological molecules involves modificationof a silica surface through the successive application of multiplelayers of biotin, avidin and extenders [see, e.q., U.S. Pat. No.4,282,287]; other methods involve photoactivation in which a polypeptidechain is attached to a solid substrate by incorporating alight-sensitive unnatural amino acid group into the polypeptide chainand exposing the product to low-energy ultraviolet light [see, e.g.,U.S. Pat. No. 4,762,881]. Oligonucleotides have also been attached usinga photochemically active reagents, such as a psoralen compound, and acoupling agent, which attaches the photoreagent to the substrate [see,e.g., U.S. Pat. No. 4,542,102 and U.S. Pat. No. 4,562,157]. Similarmethods are applicable to peptide nucleic acids. Photoactivation of thephotoreagent binds a nucleic acid molecule or peptide nucleic acidmolecule to the substrate to give a surface-bound probe. In certainembodiments, the photoactivation may occur in situ by selecting anappropriate bioluminescence generating system with an appropriateemission wavelength sufficient to photoactivate and immobilize thenucleic acid.

Furthermore, U.S. Pat. No. 5,451,683 describes a technique for attachingbiochemical ligands to surfaces of matrices by attachment of aphotoactivatable biotin derivatives. Photolytic activation of the biotinderivatives for biotin analogs having strong binding affinity for avidinor streptavidin. The biotinylated ligands are immobilized on activatedregions previously treated with avidin or streptavidin.

The attachment of anti-ligands to a matrix material may also be achievedelectronically. Self-addressable, self-assembling microelectronicsystems and devices for electronically controlling the transport andbinding of specific binding entities to specific microlocations on amatrix [e.g., see International Patent Application Publication Nos. WO95/12808; WO 96/01836 and WO 96/07917, U.S. Pat. Nos. 5,632,957,5,605,662]. Electronic control of the individual micro-locations may beeffected whereby voltage or current is controlled. When one aspect isset, the other may be monitored. For example, when voltage is set, thecurrent may be monitored. Alternativelyh, when voltage is set, thecurrent may be monitored. The voltage and/or current may be applied in adirect current mode, or may vary with time.

The spatial addressability afforded by these methods allows theformation of patterned surfaces having preselected reactivities. Forexample, by using lithographic techniques known in the semiconductorindustry, light can be directed to relatively small and precisely knownlocations on the surface. It is, therefore, possible to activatediscrete, predetermined locations on the surface for attachment ofanti-ligands. The resulting surface will have a variety of uses. Forexample, direct binding assays can be performed in which ligands can besimultaneously tested for affinity at different anti-ligands attached tothe surface.

For example, the attachment of biological molecules to a silica surfaceof the non-self addressable chip using alkoxysilanes typically involvespre-hydrolysis of the surface. All of the following operations should beperformed in a laminar flow hood/clean environment to avoidcontamination with dust, organic particles and other particulates.Typically, the appropriate alkoxysilane is dissolved in a 3:1ethanol-water solution at room temperature for 12 hours. The chip istreated by flooding a selected area of the chip repeatedly using freshaliquots of the silane-alcohol solution. After this treatment, the chipis washed using large amounts of absolute ethanol, followed by washes inTHF or dioxane, hexane (ultrapure) and finally pentane, which isevaporated under a stream of dry nitrogen.

The efficiency of the derivatization of the surface of the chip may bedetermined by coupling an appropriate fluorescent amine (carboxylderivatized) or fluorescent carboxylic acid (amino derivatized) to thesurface of the chip by exciting the fluorescence of the bound moleculesusing a laser of appropriate wavelength. Appropriate compounds for thispurpose may be amino, carboxyl or other reactive derivatives offluorescein, rhodamine or Texas Red, which are known to those of skillin the art and are also commercially available (e.g., see MolecularProbes, Inc.).

The isothiocyanates of fluorescein, rhodamine, or Texas Red, forexample, react in an irreversible and covalent manner with any freeamino groups on the silica surface. A solution of an effectiveconcentration of fluorescein (about 10 mM) isothiocyanate (mixedisomers) in acetone or dioxane is placed on the amine-derivatized silicaof the chip for sufficient time, typically about 30 minutes at ambienttemperatures. To remove all unreacted material, the chip is washed withhot (i.e., 60° C.) solutions of acetone, hexane and pentane or othersuitable solvent. A region on the same chip that has not been chemicallyderivatized is similarly treated with the fluorescein isothiocyanate asa control. A small amount of direct covalent reaction with the glass ispossible and thus the control should be performed to indicate backgroundlevels. The fluorescence of the bound fluorescein can be excited using asuitable sources, such as an argon ion laser (e.g., 488 nm), preferablyusing a 45-degree angle geometry. The argon laser can further contain aphotomultiplier equipped with a 10 nm bandpass filter for detecting theemitted fluorescence signal at about 520 nm. The amount of fluorescencedetected is a function of the extent and efficiency of derivatization.

In another embodiment, provided herein, a reflective surface, e.g.,MYLAR, may be derivatized as described above such that an anti-ligandmay be immobilized directly to the protective, activated outer surfaceoverlaying the reflective metal layer, such as a derivatized silanelayer. In this embodiment, light generated by the bioluminescencegenerating system will not be scattered or absorbed by the anti-ligandbecause the photodiodes are not occuled by bound anti-ligand.

D. Formation of Luciferase Conjugates

1. Linkers

The conjugation of a luciferase to an anti-ligand, e.g., an antibody,oligonucleotide or peptide nucleic acid, may be achieved in the absenceor presence of a linker sequence using methods known to those of skillin the art. Any linker known to those of skill in the art may be usedherein. Methods for linking a luciferase to an antibody are described inU.S. Pat. Nos. 4,657,853; 5,486,455 and International Patent ApplicationPublication No. WO 96/07100.

Other linkers are suitable for incorporation into chemically linkedproteins. Such linkers include, but are not limted to: disulfide bonds,thioether bonds, hindered disulfide bonds, and covalent bonds betweenfree reactive groups, such as amine and thiol groups. These bonds areproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the polypeptides and then reacting the thiolgroups on one polypeptide with reactive thiol groups or amine groups towhich reactive maleimido groups or thiol groups can be attached on theother. Other linkers include, acid cleavable linkers, such asbismaleimideothoxy propane; cross linkers that are cleaved upon exposureto UV or visible light. In some embodiments, several linkers may beincluded in order to take advantage of desired properties of eachlinker.

Chemical linkers and peptide linkers may be inserted by covalentlycoupling the linker to the anti ligand and to the surface of the chip.The heterobifunctional agents, described below, may be used to effectsuch covalent coupling. Peptide linkers may also be linked by expressingDNA encoding the linker and the anti ligand, e.g., an antibody, as afusion protein.

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins, are known to those of skill in this art(see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook,1992-1993, which describes the preparation of and use of such reagentsand provides a commercial source for such reagents; see, also, e.g.,Cumber et al. (1992) Bioconiugate Chem. 3:397-401; Thorpe et al. (1987)Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad Sci.84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197;Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al. (1987)Anal. Biochem. 162:163-170; Wawryznaczak et al. (1992) Br. J. Cancer66:361-366; Fattom et al. (1992) Infection & Immun. 60:584-589). Thesereagents may be used to form covalent bonds between the anti ligand andthe luciferase molcecule. These reagents include, but are not limitedto: N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; disulfidelinker); sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate(sulfo-LC-SPDP); succinimidyl-oxycarbonyl-α-methyl benzyl thiosulfate(SMBT, hindered disulfate linker); succinimidyl 6-[3-(2-pyridyidithio)propionamido]hexanoate (LC-SPDP); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-late (sulfo-SMCC);succinimidyl 3-(2-pyridyidithio)butyrate (SPDB; hindered disulfide bondlinker); sulfosuccinimidyl 2-(7-azido-4-methyl-coumarin-3-acetamide)ethyl-1,3′-dithiopropionate (SAED); sulfo-succini-midyl7-azido-4-methylcoumarin-3-acetate (SAMCA); sulfosuccinimidyl6-[alpha-methyl-alpha-(2-pyridyidithio)toluamido]hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyidithio)propionamido]butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridylthio)toluene(SMPT, hindered disulfatelinker);sulfosuccinimidyl6[α-methyl-α-(2-pyridyldithio)-toluamido]hexanoate(sulfo-LC-SM PT); m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccini-mide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl4(μ-maleimidophenyl)butyrate (SMPB);sulfo-succinimidyl4-(μ-maleimidophenyl)butyrate (sulfo-SMPB);azidobenzoyl hydrazide (ABH).

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584-589).

2. Luciferase Fusion Proteins

In addition to antibody-luciferase conjugates, a recombinant luciferaseprotein fusion to an anti ligand, e.g., an antibody or F(Ab)₂antigen-binding fragment thereof, is also contemplated for use herein.For example, the DNA encoding a monoclonal antibody may be ligated toDNA encoding a luciferase or the luciferase may be linked to an antibody[see, e.g., U.S. Pat. No. 4,478,817, which describes antibody/luciferaseconjugates and the use thereof].

3. Nucleic Acid and Peptide Nucleic Acid Conjugates

The luciferase molecules described herein may also be conjugated tonucleic acids or peptide nucleic acids. The coupling may also beeffected in the absence or presence of a linker. Methods for conjugatingnucleic acids, at the 5′ ends, 3′ ends and elsewhere, to the amino andcarboxyl terminii and other sites in proteins are known to those ofskill in the art (for a review see e.g., Goodchild, ( 1993 )In:Perspectives in Bioconiugate Chemistry, Mears, Ed., American ChemicalSociety, Washington, D.C. pp.77-99. For example, proteins have beenlinked to nucleic acids using ultraviolaet irradiation (Sperling et al.(1978) Nucleic Acids Res. 5:2755-2773; Fiser et al. (1975) FEBS Lett.52:281-283), bifunctional chemicals (Bäumert et al. (1978) Eur. J.Biochem. 89353-359; and Oste et al. (1979) Mol. Gen. Genet. 168::81-86)photochemical cross-linking (Vanin et al. (1981) FEBS Lett. 124:89-92;Rinke et al. (1980) J.Mol.Biol. 137:301-314; Millon et al. (1980) Eur.J. Biochem. 110:485-454).

In addition, the carboxyl terminus of a luciferase may be conjugated toone of the free amino groups of peptide nucleic acids [e.g., see Nielsenet al. (1990) Science 254:1497-1500; Peffer et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:10648-10652) using standard carbodiimide peptidechemistry.

Additional sites for conjugation can also be introduced into the nucleicacid molecule by chemical modification of one or more position or by theintroduction of a small antigenic determinant covalently coupled to the5′ or 3′-end of the molecule. A variety of small antigenic determinants[e.g., His Tags, fig antigens, S-Tags, dioxigenin and the like) areknown to those of skill in the art and are also commercially available[e.g., Boehringer Mannheim, Indianapolis, Ind.; Novagen, Inc., MadisonWis.]. Modified nucleic acids and peptide nucleic acid analogs may alsobe prepared by direct chemical synthesis using standard phosphoroamiditechemistry and commercially available modified nucleoside triphosphateanalogs (5′-thiolated nucleoside triphosphates and oligonucleotides). 5′and 3′ thiolated oligonucleotides are also commercially available [e.g.,Operon Technologies, Alameda, Calif.].

E. Radiolaria and Diatoms for Depositing Silica on Matrices

A method of using biomineralization to deposit silica on a matrixmaterial is also provided herein. The method uses diatom and radiolariaenzymes and cell wall proteins to effect the polymerization of silicondioxide along the interface region of the matrix to form amatrix-silicate mesostructure. This method may be used in thesemiconductor industry for the preparation of silicate chips that have avariety of end use applications.

Organisms such as diatoms and radiolaria synthesize elaborate biomineralsilica-based cell walls, also termed frustulum/frustles or exoskeletons,which display hierarchichal structures patterned on scales from lessthat a micrometer to millimeters [e.g., see in general, Anderson (1983)in Radiolaria, Spriner-Verlag, N.Y.; Sullivan (1986) Ciba Found. Symp.121:59-89]. The two main principles of the architecture in diatom cellwalls are cell walls with radial symmetry (centric diatoms; e.g.,Cylindrotheca crypta) and those with bilateral symmetry (pennatediatoms; e.g., Navicula peliculosa and C. fusiformis).

The diatom cell wall includes of two parts, the epitheca and thehypotheca. Each theca is composed of a valve and several silica strips,girdle bands, which are composed of amorphous, hydrated silica and otherorganic components [e.g., see Volcani (1981) in Silicon and SiliceousStructures in Biological Systems, Simpson and Volcani, eds, pp. 157-200,Springer-Verlag; Kroger et al. (1996) EMBO 13:4676-4683]. The majororganic protein constituents of these cell walls is a family of proteinsknown as frustulins [see, e.g., Kroger et al. (1996) Eur. J. Biochem.239:259-264]. In marine diatoms, new valves are produced after celldivision and cytokinesis of the mother protoplast. The resultingdaughter protoplast produces a new valve in a specialized intracellularorganelle, the silica deposition vesicle. Silica is transported into thesilicalemma where nucleation and epitaxial growth of Si monomers occurson a template or more complex polymerization of silica occurs within thevesicles [see, e.g., Pickett-Heaps et al. (1979) Bio. Cell. 35:199-203;Sullivan (1986) Ciba Found. Symp. 121:59-89; Pickett-Heaps et al. (1990)Prog. Phycol. Res. 7:1-186].

In radiolaria, the deposition of the silicate skeleton is associatedwith a cytoplasmic sheath that encloses, molds and deposits the skeletontermed “cytokalymma”. The thickness of the skeleton may be influenced bythe physiological state of the organism. The cytokalymma may function inan analogous manner to the silicalemma in the silica deposition indiatoms.

Artifical inorganic assemblies that mimic diatom and radiolariaexoskeletons have been described [e.g., see Oliver et al. (1995) Nature378:47-50; U.S. Pat. Nos. 5,057,296, 5,108,725 and 5,364,797]. Severalmorphologies of mesophases may be formed, e.g., lamellar, hexagonal andcubic mesostructures, depending on the selected starting materials andconditions used. These crystalline mesostructures, however, may only beformed at higher temperatures, which may be unsuitable for use withcertain matrix materials.

Models have been proposed to explain the biomineralization process andalso the formation and morphology of these surfactant-silicatemesostructures [see, e.g., Sullivan (Monnier et al. Science261:1299-1303]. For example, it is postulated that the control of thesilicate wall thickness is related to the double layer potential:silicate species only accumulate at the surface interface, which wouldthicken the wall or produce amorphous bulk SiO₂, does not occur becauseof the strong electrostatic replusion produced by the high negativecharge on the silicate species at the high pHs at which these areformed, e.g., pH 12 and above [e.g., llier (1979) in The Chemistry ofSilica, p. 182, Wiley, N.Y.].

Artificial assemblies of mesoprous crystalline material containing M41Shas been included in sensor sevices, including biosensors [see, e.g.,U.S. Pat. No. 5,364,797]. In biosensors, either biological analyte ineach pair is affixed to the ultra-large pore crystalline substrate bycovalent binding to silanols in the crystalline material [e.g., Harlowet al. Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]. The analyte, e.g., an antibody, is attached and the interactionbetween the affixed analyte and the test sample is monitored.

The diatom silica-based cell walls are analogous in structure to the theabove-described artificial assemblies. Thus, these silica deposits mayhave similar morphology and optical properties as the fiber opticsensors to the M41S artificial assemblies useful in biosensors and othermolecular biological apparatus [see, e.g., U.S. Pat. No. 5,364,797].

In a method of using biomineralization using the enzymes and cell wallproteins of the silicalemma and/or silica deposition vesicles of diatomsto deposit silica on the surface of a matrix material of a chip isprovided herein, silica may be deposited on a matrix material that hasbeen linked with a uniform coating of a Si template using a silicalemmaisolated from a diatom. The deposited silica may be attached to thematrix through another suitable linker, such as sugars or otherdiol-containing compounds. Alternatively, the components of thesilicalemma may be further purified using methods known to those ofskill in the art in protein chemistry.

F. Methods of Use

1. Immunoassays

The chips described herein may be used in diagnostic assays. Forexample, the chips are used in an immunosandwich assay for the detectionof infectious agents using antibodies directed against ainfectiousmicroorganisms, e.g., bacteria, viruses, protozoa and other lowereukaryotic organisms [e.g., see FIG. 20]. A plurality of anti-ligands,e.g., antibodies, is linked to each location or microlocation on thechip or attached to an appropriate layer of the reflective middle layerof a multi-well chip creating a panel of antibodies raised against aparticular microorganisms. The antibody-bound chip is placed directlyinto a sample of body fluid obtained from a patient, e.g., urine, sputumor blood.

Sufficient time is allowed to form antibody-antigen complexes and thechip is removed and rinsed thoroughly. A solution containing a pluralityof secondary antibodies directed against a panel of known pathogensconjugated to a luciferase or luciferase fusion protein is added, whichmay be directed against the same antigen or another antigen present onthe targeted species. Alternatively, a phage or virus may be employedthat has been genetically engineered to contain DNA encoding aluciferase. Preferably, the virus or phage has a broad specificity.

The chip or individual well is washed, and the remaining components ofthe bioluminescence generating system, e.g., a luciferin and anynecessary activators, are added. If an antigen has been detected, lightis emitted from the bound luciferase, which is in turn detected by thephotodiodes located within the semiconductor layer in the attached chip.In the multi-well chip system, the output signal of the bioluminescentreaction is increased by detecting light directly emitted from thereaction as well as light reflected off of the middle layer [e.g., seeFIGS. 10 & 11]. The output signal may optionally be amplified and/ormultiplexed prior to be sent to a computer processing unit for dataanalysis.

The assay may be used quantitatively by adding a known amount ofluciferin to the well and by measuring the rate of the utilization ofthe luciferin (i.e., a reduction in light production over time isproportional to the amount present in the sample as compared tocontrols).

2. Nucleic Acid Hybridization Assays

The chips described herein may also be used in nucleic acidhybridization assays. For example, a desired nucleic acid or peptidenucleic acid probe or a nucleic acid with linked peptide is covalentlycoupled to the derivatized silica surface of the chip directly or via alinker group. The nucleic acids can be coupled to the entire surface orthe chip or may be added to one or more microlocations on the chip in anarray format.

The infectious agents present in the biological sample are lysed usingchemical, enzymatic or physical means and the nucleic acids, preferablyDNA, is isolated from the sample using standard methods known to thoseof skill in the art [e.g., see Sambrook et al., (1989) MolecularCloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York].Alternatively, the sample can be analyzed without purification of thenucleic acid species.

The nucleic acid is resuspended in hybridization buffer and the sampleis added to the surface of the chip and incubated at the desiredhybridization temperature. After allowing sufficient time forhybridization, the chip is washed thoroughly and then washed under theappropriate stringency conditions as described herein, i.e. high mediumor low. The complementary nucleic acid immobilized to the chip isdetected by the addition of an anti ligand conjugated to a component ofa bioluminescence generating system, preferably a luciferase. Presentlypreferred anti ligands are antibodies, or a F(Ab)₂ fragments thereof,that preferentially recognize double stranded nucleic acids or theassociated small antigenic determinant. Antibodies that recognize doublestranded DNA are associated with a number of autoimmune diseases [e.g.,see Tsuzaka et al. (1996) Clin. Exp. Immunol. 106:504-508; Kanda et al.(1997) Arthritis Rhem. 40:1703-1711.

The chip or individual well is washed to remove unbound antibodyluciferase conjugate, and the remaining components of thebioluminescence generating system, e.g., a luciferin and any necessaryactivators, are added. If a complementary nucleic acid or peptidenucleic acid has been detected, light is emitted from the boundluciferase, which is in turn detected by the photodiodes located withinthe semiconductor layer in the attached chip.

The assay may also be used quantitatively by adding a known amount ofluciferin to the well and by measuring the rate of the utilization ofthe luciferin (i.e., a reduction in light production over time isproportional to the amount present in the sample as compared tocontrols).

3. Detection of Antibiotic Sensitivity

Among other uses for the chip is testing the sensitivity of a clinicalisolate to known antibiotics or as a device to screen for antibacterialagents. For example, after detecting light emission from a targetedwell, an isolate may be grown directly in the well for a short period bythe addition of a suitable growth medium [e.g., L-broth or otherundefined medium] followed by incubation under appropriate enviromentalconditions, such as temperatures of 20° C. to 42° C. under aerobic oranaerobic atmospheres.

The growing bacteria are then infected with a bacteriophage, such aslambda or P22 for enterobacteria, that has been genetically engineeredto encode firefly luciferase, which requires available ATP as aco-factor [see e.g., Section B.4]. The expression of intracellularluciferase in these bacteria, in the presence of ATP, results in theproduction light.

The effectiveness of antibiotic therapy can be monitored directly inthis system by incubating the bacteria with an effective concentrationof an antibiotic and following subsequent light emission. If theantibiotic results in cell death, intracellular ATP pools will bedepleted thereby inhibiting the bioluminescent reaction. The decrease inlight is suggestive that the particular antibiotic or compound iseffective. In other embodiments, the bacterial are incubated with testcompounds and the antibacterial activity of the test compound isassessed.

4. Synthetic Synapse

Versions of the chips provided herein may also be used to generate asynthetic neuronal synapse [e.g., see FIGS. 17-19]. A suitable enzyme,particularly, acetycholine esterase is fused to a luciferase, such as byrecombinant expression. The luciferase is either in an inactive oractive conformation. Suitable mutations in either protein may beselected to insure that luciferase can undergo appropriateconformational changes as described herein. The resulting fusion isattached to a chip, such as a chip provided herein. The neuron or bundleof neurons is kept in close proximity to the fusion protein linked tothe chip by providing neuronal growth factors, e.g., EGF or NGF, nearthe location of the chip through a microport to promote and maintainlocal neurite outgrowth [see FIG. 17].

The silicon-synapse electrodes may be permenantly implanted in anafflicted patient by insertion into the appropriate stereotaxiclocations in the spinal cord by MRI localization [see FIG. 19]. Toimplant the electrodes, microholes are drilled into the spinal cordusing a suitable laser, such as a CO₂ laser, and the electrode is placedinto proximity of a known nerve fiber or bundle. The placement of thesilicon-synapse may be from superficial to deep within the spinal cordalong known neuronal pathways. Exact tracing of the appropriate neuronis preferable, though not essential, because the human brain willreprogram itself to send the signal along those neurons that transmitthe proper signal.

The transmission of neuronal impulses involves variousneurotransmitters, such as acetylcholine, which are released into thesynapse. Upon binding of the ligand to the enzyme, such as the bindingof acetylcholine to the esterase, the linked luciferase is, ifpreviously inactive, is activated by the binding, or if previouslyactive, is inactivated by the binding [see FIG. 18]. In the presence ofthe remaining components of a bioluminescence generating system, lightis produced (or is quenched), which change is detected by thephotodiodes associated with the chip. This detection generates one ormore electrical or data signals that is/are sent through one or morewires leading to a computer, such a miniature computer that is attachedto a belt, which processes the information. The processed information istransmitted by appropriate means, such as a fiber, to one or moreelectrodes, which are attached to any desired device or effector,particularly a muscle. Upon receipt of the signal, work, such as amuscle twitch, occurs and body movements may be initiated. The deviceswill be inserted in a manner that bypasses a lesioned area of the spinalcord [see, e.g., see FIG. 17].

Alternatively, the acetylcholine binding region of acetylcholineesterase may be fused to a fluorochrome or phycobiliprotein and used inconjunction with a laser. In this embodiment, monochromatic light of aknown wavelength is generated by a laser to excite the fluorophore andthe emitted fluorescence is directed to the photodiode surface of thechip by a parabolic mirror [see e.g., FIGS. 17 & 18], and the emittedlight detected and employed as described for the bioluminescence.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

Summary of Sequences of Representative Luciferases and the Reductase SetForth in the Sequence Listing

1. SEQ ID NO. 1 Renilla reinformis Luciferase [U.S. Pat. No. 5,418,155]

2. SEQ ID NO. 2 Cypridina hilgendorfii luciferase [EP 0 387 355]

3. SEQ ID NO. 3 Modified Luciola cruciata Luciferase [firefly; U.S. Pat.No. 4,968,613]

4. SEQ ID NO. 4 Vargula (Cypridina) luciferase [Thompson et al. (1989)Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571 and from JP 3-30678 Osaka

5. SEQ ID NO. 5 Apoaequorin-encoding gene [U S. Pat. No. 5,093,240,pAQ440]

6. SEQ ID NO. 6 Recombinant Aequorin AEQ1 [Prasher et al. (1987)“Sequence Comparisons of cDNAs Encoding for Aequorin Isotypes,”Biochemistry 26:1326-1332]

7. SEQ ID NO. 7 Recombinant Aequorin AEQ2 [Prasher et al. (1987)]

8. SEQ ID NO. 8 Recombinant Aequorin AEQ3 [Prasher et al. (1987)]

9. SEQ ID NO. 9 Aequorin photoprotein [Charbonneau et al. (1985) “AminoAcid Sequence of the Calcium-Dependent Photoprotein Aequorin,”Biochemistry 24:6762-6771]

10. SEQ ID NO. 10 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728; Asp 124 changed to Ser]

11. SEQ ID NO. 11 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728; Glu 135 changed to Ser]

12. SEQ ID NO. 12 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728 Gly 129 changed to Ala]

13. SEQ ID NO. 13 Recombinant apoaequorin [sold by Sealite, Sciences,Bogart, Ga. as AQUALITE®, when reconstituted to form aequorin]

14. SEQ ID NO. 14 Vibrio fisheri Flavin reductase [U.S. Pat. No.5,484,723].

We claim:
 1. A microelectronic device, comprising: a substrate; aplurality of micro-locations defined on the substrate, wherein eachmicro-location is for linking a macromolecule; an independentphotodetector integrated at each micro-location and optically coupled toeach micro-location, each photodetector being configured to generate asensed signal responsive to the photons of light emitted at thecorresponding micro-location when a light-emitting chemical reactionoccurs at that micro-location, each photodetector being independent fromthe photodetectors optically coupled to the other micro-locations; andan electronic circuit coupled to each photodetector and configured toread the sensed signal generated by each photodetector and to generateoutput data signals therefrom that are indicative of the light emittedat each micro-location by the light-emitting chemical reactions, wherebythe device detects photons of light emitted by light-emitting chemicalreactions, wherein each micro-location is defined by a portion of thesurface of the device.
 2. The device of claim 1, wherein themicro-locations are derivatized for linking proteins, nucleic acids ororganic molecules.
 3. The device of claim 1, further comprising linkedmacromolecules.
 4. The device of claim 1, wherein the micro-locationsare provided as an array. component of a bioluminescence generatingsystem.
 5. The microelectronic device of claim 1, wherein themicro-locations defmed on the substrate each comprise a chemicalreactant that emits photons of light when a reaction takes place at thatmicro-location.
 6. The device of claim 5, wherein the chemical reactantis a component of a bioluminescence generating system.
 7. The device ofclaim 6, wherein the component of a bioluminescence generating system isa luciferase or luciferin.
 8. The device of claim 6, wherein thecomponent of a bioluminescence generating system is a luciferase that isa photoprotein.
 9. The device of claim 6, wherein the bioluminescencegenerating system is selected from the group consisting of Aequorea,Vargula, Renilla, Obelin, Porichthys, Odontosyllis, Aristostomias,Pachystomias, firefly, and bacterial bioluminescence generating systems.10. The microelectronic device of claim 1, wherein: the substrate is asemiconductor substrate, comprising a surface that is adapted forlinking macromolecules: each micro-location is defined by a portion ofthe surface that is adapted to allow a separate chemical reactant atthat micro-location to be coupled thereto.
 11. The device of claim 10,wherein the surface is coated with an inert material that is derivatizedfor linking macromolecules.
 12. The microelectronic device of claim 1,wherein the electronic circuit includes a pixel unit cell circuitassociated with each photodiode and a delta-sigma A/D conversioncircuit, each pixel unit cell circuit being configured to integrate thesensed signal from the respective photodiode and the A/D conversioncircuit being configured to quantize the integrated sensed signals. 13.The microelectronic device of claim 12, wherein each pixel unit cellcircuit is addressable and the electronic circuit further includes anaddress control circuit for sequentially addressing each pixel unit cellcircuit, and wherein the A/D conversion circuit quantizes the integratedsensed signal of the pixel unit cell circuit being addressed by theaddress control circuit.
 14. The microelectronic device of claim 13,wherein each photodiode converts photons of light emitted by thechemical reaction into a photocurrent comprising a magnitude dependingon the number of photons, and each pixel unit cell circuit includes acapacitance circuit comprising a charge that changes at a rate dependenton the magnitude of the photocurrent, whereby the sensed signal isintegrated by the capacitance circuit.
 15. The microelectronic device ofclaim 14, wherein each pixel unit cell circuit generates an outputcurrent that depends on the charge of the capacitance circuit when thepixel unit cell circuit is addressed, the electronic circuit alsoincluding a comparator circuit for comparing the output current of theaddressed pixel unit cell circuit to a reference current to generate afeedback signal used to reset the capacitance circuit to an initialcharge when the output current transitions with respect to the referencecurrent.
 16. The microelectronic device of claim 15, wherein theelectronic circuit further includes an output control circuit thatreceives the feedback signal from each addressed pixel unit cellcircuit, and generates the output data signals as a serial output datastream based upon the feedback signals, the rate of feedback signaltransitions correlated with each micro-location being indicative of theemitted light at that micro-location.
 17. The device of claim 1, furthercomprising a layer of reflective material on all or a portion of thesurface of the device or above the surface of the device, whereby lightgenerated in the reaction is reflected thereby enhancing the lightsignal detected by the photodetector.
 18. The device of claim 17,therein the material is oriented polyethylene terephthalate.
 19. Themicroelectronic device of claim 1, wherein: the micro-locations definedon the substrate are for receiving a fluid sample for analysis, eachmicro-location comprising an attachment layer to which macromoleculesare linked; a macromolecule is linked to each of a plurality of themicro-locations via the attachment layer; a linked macromoleculeselectively binds to analyte present in the sample received by thedevice; and each photodetector is configured to generate a sensed signalresponsive to photons of light emitted at the correspondingmicro-location when the selected analyte bound at that micro-location isexposed to a second macromolecule that binds to the first macromoleculeor analyte linked to one or more components of a light-emitting reactionin the presence of the remaining components of the light-emittingreaction.
 20. The device of claim 19, wherein each macromolecule is anantibody and the analyte is an antigen.
 21. The device of claim 19,wherein the micro-locations defined on the substrate for receiving thefluid sample to be analyzed form wells in the surface of the device. 22.The device of claim 21, wherein one or a plurality of the wells comprisea reflective material disposed along the sides thereof or suspendedacross the well, whereby light is reflected to the photodetector. 23.The device of claim 19, comprising a layer of reflective material on allor a portion of micro-locations defined on the substrate that are forreceiving a fluid sample.
 24. The device of claim 23, wherein thereflective material is oriented polyethylene terephthalate.
 25. Thedevice of claim 19, wherein the light-emitting reaction is luminescence.26. The device of claim 25, wherein the luminescence is bioluminescence.27. The device of claim 19, further comprising at least one component ofa bioluminescence generating system in each micro-location thatcomprises a macromolecule.
 28. The device of claim 1, wherein thephotodetector optically coupled to each micro-location is configured togenerate a sensed signal responsive to bioluminescence emitted at thecorresponding micro-location.
 29. The device of claim 19, wherein thephotodetector optically coupled to each micro-location is configured togenerate a sensed signal responsive to bioluminescence emitted at thecorresponding micro-location.
 30. The device of claim 26, wherein thebioluminescence is produced by a bioluminescence generating system thatcomprises a luciferase or luciferin.
 31. The device of claim 30, whereinthe luciferase is a photoprotein.
 32. The device of claim 26, whereinthe bioluminescence is produced by a bioluminescence generating systemthat is selected from the group consisting of the Aequorea, Vargula,Renilla, Obelin, Porichthys, Odontosyllis, Aristostomias, Pachystomias,firefly, and bacterial bioluminescence generating systems.
 33. Themicroelectronic device of claim 19, wherein the device comprises aplurality of different macromolecules each specific for a differentanalyte, each different macromolecule present at a differentmicro-location.
 34. The microelectronic device of claim 19, wherein: themicro-locations are in the form of an array; the array ofmicro-locations includes a first and a second array of pixel elementscomprising a first and a second size, respectively, the first and secondsizes being different.
 35. The microelectronic device of claim 34,wherein the macromolecule attached to the attachment layer of the firstpixel element array is a receptor antibody that specifically binds afirst selected analyte and the macromolecule attached to the attachmentlayer of the second pixel element array is a receptor antibody thatspecifically binds a second selected analyte different from the firstselected analyte.
 36. The microelectronic device of claim 19, whereinthe substrate is semiconductor and each micro-location is located on asurface of the semiconductor substrate, with the surface at eachmicro-location defining the attachment layer for that micro-location.37. The microelectronic device of claim 36, wherein: the surface of thesemiconductor substrate is derivatized to enhance the attachment of themacromolecule to the attachment layer at each micro-location; and saidmacromolecule is a receptor antibody.
 38. The microelectronic device ofclaim 37, wherein each photodetector includes a photodiode located atthe surface of the respective micro-location, and the reaction producesphotons of light converted by the photodiode into a photocurrent whenthe selected analyte is present in the sample, the photocurrent beingthe sensed signal generated by the photodiode.
 39. The microelectronicdevice of claim 38, wherein the electronic circuit includes a pixel unitcell circuit associated with each photodiode and a delta-sigma A/Dconversion circuit, each pixel unit cell circuit being configured tointegrate the sensed signal from the respective photodiode and the A/Dconversion circuit being configured to quantize the integrated sensedsignals.
 40. The microelectronic device of claim 39, wherein each pixelunit cell is addressable and the electronic circuit further includes anaddress control circuit for sequentially addressing each pixel unitcell, and wherein the A/D conversion circuit quantizes the integratedsensed signal of the pixel unit cell circuit being addressed by theaddress control circuit.
 41. The microelectronic device of claim 38,wherein: photons of light are generated by a bioluminescence generatingsystem that comprises a luciferase and a luciferin; each photodiodeconverts photons of light emitted by the bioluminescence generatingsystem into a photocurrent comprising a magnitude that depends on theconcentration of the selected analyte in the sample, and each pixel unitcell circuit includes a capacitance circuit comprising a charge thatchanges at a rate dependent on the magnitude of the photocurrent,whereby the sensed signal is integrated.
 42. The microelectronic deviceof claim 41, wherein each pixel unit cell circuit generates an outputcurrent that depends on the charge of the capacitance circuit when thepixel unit cell circuit is addressed, the electronic circuit alsoincluding a comparator circuit for comparing the output current of theaddressed pixel unit cell circuit to a reference current to generate afeedback signal used to reset the capacitance circuit to an initialcharge when the output current transitions with respect to the referencecurrent.
 43. The microelectronic circuit of claim 42, wherein theelectronic circuit further includes an output control circuit thatreceives the feedback signal from each addressed pixel unit cellcircuit, and generates the output data signals as a serial output datastream based upon the feedback signals, the rate of feedback signaltransitions correlated with each micro-location being indicative of thebioluminescence emitted at that micro-location.
 44. A system fordetecting and identifying an analytes in a biological sample usingluciferase-luciferin bioluminescence, comprising: the microelectronicdevice of claim 1 wherein light emitted at the correspondingmicro-location is bioluminescence; a processing instrument including: aninput interface circuit coupled to the microelectronic device forreceiving the output data signals indicative of the bioluminescenceemitted at each micro-location; a memory circuit for storing a dataacquisition array comprising a location associated with eachmicro-location; an output device for generating visible indicia inresponse to an output device signal; and a processing circuit coupled tothe input interface circuit, the memory circuit, and the output device,the processing circuit being configured to read the output data signalsreceived by the input interface circuit, to correlate the output datasignals with the corresponding micro-locations, to integrate the outputdata signals correlated with each micro-location for a desired timeperiod by accumulating the output data signals in the data acquisitionarray, and to generate the output device signal which, when applied tothe output device, causes the output device to generate visible indiciarelated to the presence of the selected analytes in the sample.
 45. Thesystem of claim 44, wherein the microelectronic device comprises: anarray of micro-locations for receiving the biological sample to beanalyzed, each micro-location comprising an attachment layer; a separateantibody attached to the attachment layer of each micro-location, eachantibody specific for binding a selected analyte present in the samplereceived by the array; a photodetector optically coupled to eachmicro-location, each photodetector being configured to generate a sensedsignal responsive to bio-luminescence emitted at the correspondingmicro-location; and an electronic circuit coupled to each photodetectorand configured to read the sensed signal generated by each photodetectorand generate output data signals therefrom that are indicative of thebioluminescence emitted at each micro-location by theluciferase-luciferin reaction.
 46. The system of claim 45, wherein; eachmicro-location is located on a surface of a semiconductor substrate andeach photodetector includes a photodiode located on the surface at therespective micro-location; the bioluminescence generating reactionproducing photons of light that are converted by the photodiode into aphotocurrent when the selected analyte is present; and the photocurrentis a sensed signal generated by the photodiode.
 47. The system of claim45, wherein the electronic circuit of the microelectronic deviceincludes an output control circuit that generates a serial data streamcomprising the output data signals, and the input interface circuit ofthe processing instrument includes a serial interface circuit configuredto receive the serial data stream from the microelectronic device. 48.The system of claim 47, wherein the serial data stream includesmultiplexed data representative of the bioluminescence emitted at eachmicro-location by the luciferase-luciferin reaction.
 49. The system ofclaim 45, wherein the output device includes an electronic display, andthe visible indicia includes light emitted by the display.
 50. Thesystem of claim 45, wherein the memory circuit also stores an analytemap identifying the selected analyte at each micro-location, and theprocessing circuit correlates the integrated output data signals in thedata acquisition array with the analyte map to identify the selectedanalytes present in the sample, the output device signal being generatedsuch that the visible indicia identifies the selected analytes presentin the sample.
 51. The system of claim 45, wherein the processinginstrument further comprises an input device coupled to the processingcircuit for generating desired integration time period signals used bythe processing circuit to determine the desired integration time periodfor the output data signals.
 52. The system of claims 45, wherein theboluminescence generating system is selected from the group consistingof those isolated from the ctenophores, coelenterases, molluscan fish,ostracods, insects, bacteria, crustacea, annelids and earthworms. 53.The system of claim 45, wherein the component of the bioluminescencegenerating system linked to the macromolecule is selected from the groupconsisting of Aequorea, Vargula, Renilla, Obelin, Porichthys,Odontosyllis, Aristostomias, Pachystomias, firefly, and bacterialbioluminescence generating systems.
 54. The system of claim 45, hereinthe antibody attached to the attachment layer at a first micro-locationis specific for binding a first selected analyte and the antibodyattached to the attachment layer at a second micro-location is specificfor binding a second selected analyte different from the first selectedanalyte.
 55. The device of claim 6, wherein a component of thebioluminescence generating system is selected from the group consistingof bacterial, mushroom, dinoflagellate, coelenterate, ctenophore,annelid, crustacea, ostracod, copepods, insect, oleopterid, diptera,echinoderm, chordate ticate and fish bioluminescence generating systems.56. The device of claim 6, wherein a component of the bioluminescencegenerating system is selected from the group consisting of brittle star,sea cucumber, cartilaginous, bony fish, ponyfish, flashlight fish,angler fish, midshipman fish, midwater fish, marine polychaetes, syllidfireworm, jellyfish, hydroid, sea pansy, earthworm, mollusc, limpet,deep-sea fish, clam, firefly, click beetle, railroad worms and squidbioluminescence generating systems.
 57. The device of claim 6, wherein acomponent of the bioluminescence generating system is selected from thegroup consisting of Aequorea, Vargula, Renilla, Obelin, Porichthys,Odontosyllis, Aristostomias, Pachystomias, Gonadostomias, Gaussia,Halisturia, Vampire squid, Glyphus, Mycotophids, Vinciguerria, Howella,Florenciella, Chaudiodus, Melanocostus, Paracanthus, Atolla, Pelagia,Pitilocarpus, Acanthophyra, Siphonophore, Periphylla, Cavarnularia,Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus and Sea Pens,Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia, cuttlefish, andSepiolina.
 58. The device of claim 6, wherein the bioluminescencegenerating system is selected from the group consisting of theGonadostomias, Gaussia, Halisturia, Vampire squid, Glyphus, Mycotophids(fish), Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostus,Paracanthus, Atolla, Pelagia, Pitilosarcus, Acanthophyra, Siphonophore,Periphylla and Sea Pens (Stylata) bioluminescence generating systems.59. The device of claim 6, wherein the bioluminescence generating systemis selected from the group consisting of a marine, terrestrial orbacterial bioluminescence generating systems.
 60. The device of claim 6,wherein the bioluminescence generating system is selected from the groupconsisting of fungal, algal, dinoflagellate, arthropod, mollusk,echinoderm, chordate and annelid bioluminescence generating systems. 61.The device of claim 1, further comprising a layer of reflective materialon all or a portion of micro-locations defined on the substrate.
 62. Amicroelectronic device, comprising: a substrate; a plurality ofmicro-locations defined on the substrate, wherein each micro-location isfor linking a macromolecule and is defined by a portion of the surfaceof the device; an independent photodetector integrated at eachmicro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emnitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations; and an electronic circuit coupledto each photodetector and configured to read the sensed signal generatedby each photodetector and to generate output data signals therefrom thatare indicative of the light emitted at each micro-location by thelight-emitting chemical reactions, whereby the device detects photons oflight emitted by light-emnitting chemical reactions, wherein theelectronic circuit includes a pixel unit cell circuit associated witheach photodiode and a delta-sigma A/D conversion circuit, each pixelunit cell circuit being configured to integrate the sensed signal fromthe respective photodiode and the A/D conversion circuit beingconfigured to quantize the integrated sensed signals.
 63. Amicroelectronic device, comprising: a substrate; a plurality ofmicro-locations defined on the substrate, wherein each micro-location isfor linking a macromolecule and is defined by a portion of the surfaceof the device; an independent photodetector integrated at eachmicro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations; a layer of polyethyleneterephthalate reflective material on all or a portion on the surface ofthe device or above the surface of the device, whereby light generatedin the reaction is reflected thereby enhancing the light signal detectedby the photodetector; and an electronic circuit coupled to eachphotodetector and configured to read the sensed signal generated by eachphotodetector and to generate output data signals therefrom that areindicative of the light emitted at each micro-location by thelight-emitting chemical reactions, whereby the device detects photons oflight emitted by light-emitting chemical reactions.
 64. Amicroelectronic device, comprising: a substrate; a plurality ofmicro-locations defined on the substrate, wherein each micro-location isfor linking a macromolecule and is defined by a portion of the surfaceof the device; an independent photodetector integrated at eachmicro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations, wherein the photodetectoroptically coupled to each micro-location is configured to generate asensed signal responsive to bioluminescence emitted at the correspondingmicro-location; and an electronic circuit coupled to each photodetectorand configured to read the sensed signal generated by each photodetectorand to generate output data signals therefrom that are indicative of thelight emitted at each micro-location by the light-emitting chemicalreactions, whereby the device detects photons of light emitted bylight-emitting chemical reactions.
 65. A microelectronic device,comprising: a substrate; a plurality of micro-locations defined on thesubstrate, wherein each micro-location is for linking a macromoleculeand the micro-locations are in the form of an array, the array ofmicro-locations including a first and a second array of pixel elementscomprising a first size and a second size, respectively, the first andsecond sizes being different; an independent photodetector integrated ateach micro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations; and an electronic circuit coupledto each photodetector and configured to read the sensed signal generatedby each photodetector and to generate output data signals therefrom thatare indicative of the light emitted at each micro-location by thelight-emitting chemical reactions, whereby the device detects photons oflight emitted by light-emitting chemical reactions, wherein: themicro-locations defined on the substrate are for receiving a fluidsample for analysis, each micro-location comprising an attachment layerto which macromolecules are linked; a macromolecule is linked to each ofa plurality of the micro-locations via the attachment layer; a linkedmacromolecule selectively binds to analyte present in the samplereceived by the device; and each photodetector is configured to generatea sensed signal responsive to photons of light emitted at thecorresponding mnicro-location when the selected analyte bound at thatmicro-location is exposed to a second macromolecule that binds to thefirst macromolecule or analyte linked to one or more components of alight-emitting reaction in the presence of the remaining components ofthe light-emitting reaction.
 66. A microelectronic device, comprising: asubstrate; a plurality of micro-locations defined on the substrate,wherein each micro-location is for linking a macromolecule and isdefined by a portion of the surface of the device; a layer of reflectivematerial on all or a portion of the micro-portions defined on thesubstrate; an independent photodetector integrated at eachmicro-location and optically coupled to each micro-location, eachphotodetector being configured to generate a sensed signal responsive tothe photons of light emitted at the corresponding micro-location when alight-emitting chemical reaction occurs at that micro-location, eachphotodetector being independent from the photodetectors opticallycoupled to the other micro-locations; and an electronic circuit coupledto each photodetector and configured to read the sensed signal generatedby each photodetector and to generate output data signals therefrom thatare indicative of the light emitted at each micro-location by thelight-emitting chemical reactions, whereby the device detects photons oflight emitted by light-emitting chemical reactions.